Flow Field-flow Fractionation Channel Research Articles

Flow field-flow fractionation for hydrodynamic diameter estimation of gold nanoparticles with various types of surface coatings.

Flow field-flow fractionation (FlFFF) with inductively coupled plasma mass spectrometric (ICP-MS) detection was applied for estimating the hydrodynamic diameter of gold nanoparticles (AuNPs). Hydrodynamic diameters of AuNPs of the same core diameter but with different surface coatings were different because the coating agents and their properties were different. The challenge of this work is due to the fact that AuNPs with various types of surface coatings exhibited different interactions in the FlFFF channel, leading to different retention behaviors. Therefore, we are interested in finding suitable FlFFF conditions for estimating the hydrodynamic diameter of AuNPs with various types of electrostatic stabilizing agents [tannic acid (TA) and citrate (CT)] and steric stabilizing agents [polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and branched polyethylene imine (BPEI)]. Different types of carrier liquids (DI water, 0.02% FL-70, 0.05% SDS, and 30mM Tris buffer) and membrane materials [regenerated cellulose (RC) and polyethersulfone (PES) membranes] were investigated. Generally, FlFFF was applied for size characterization of nanoparticles based on FlFFF theory but the interactions between AuNPs and membrane affected the retention and the experimentally obtained hydrodynamic diameters of AuNPs from the FlFFF system. With DI water as a carrier liquid with RC or PES membranes, the hydrodynamic diameters of negatively charged particles (TA-, CT-, PVP-, and PEG-stabilized AuNPs) from FlFFF corresponded well with the hydrodynamic diameters from dynamic light scattering (DLS). Interestingly, it was possible to estimate hydrodynamic diameters of AuNPs in the mixture by using FlFFF whereas it was not possible with the use of DLS within the size range studied. This work summarized the possible interactions between AuNPs with various coating agents and membrane materials in different carrier liquids to give guidelines on the suitable conditions of FlFFF for further applications on AuNP hydrodynamic diameter estimation.

Membranes for separation of biomacromolecules and bioparticles via flow field-flow fractionation

Flow field-flow fractionation (FlFFF) is a liquid-phase separation technique that can separate macromolecules and particles on the basis of differences in their diffusion coefficient, and therefore their size. The membrane in the FlFFF channel is one of the most important elements involved in accomplishing the separation. This mini-review focuses on requirements and important points related to membranes used in FlFFF channels. Unless it is selected properly, the membrane can be a weak point of the technique due to risks such as sample loss because of the interactions between the sample and the membrane. © 2014 Society of Chemical Industry

Chip-Type Asymmetrical Flow Field-Flow Fractionation Channel Coupled with Mass Spectrometry for Top-Down Protein Identification

A chip-type design asymmetrical flow field-flow fractionation (AF4) channel has been developed for high-speed separation of proteins and top-down proteomic analysis using online coupled electrospray ionization mass spectrometry (ESI-MS). The new miniaturized AF4 channel was assembled by stacking multilayer thin stainless steel (SS, 1.5 mm each) plates embedded with an SS frit in such a way that the total thickness of the channel assembly was about 6 mm. The efficiency of the miniaturized AF4 channel at different channel lengths was examined with the separation of protein standards by adjusting flow rates in which an identical effective channel flow rate or an identical void time can be maintained at different channels. Detection limit, overloading effect, reproducibility, and influence of channel membrane materials on separation efficiency were investigated. Desalting and purification of proteins achieved during the AF4 operation by the action of an exiting crossflow and the use of aqueous mass-spectrometry-compatible (MS-compatible) buffer were advantageous for online coupling of the chip-type AF4 with ESI-MS. The direct coupling of AF4 and ESI-MS capabilities was demonstrated for the high-speed separation and identification of carbonic anhydrase (29 kDa) and transferrin (78 kDa) by full scan MS and for the first top-down identification of proteins with AF4-ESI-MS-MS using collision-induced fragmentation (CID). The presence of intact dimers (156 kDa) of transferrin was confirmed by AF4-ESI-MS via size separation of the dimers from monomers, followed by multiply charged ion spectral analysis of the dimers and molecular mass determinations. It was also found from these experiments that AF4-ESI-MS analysis of transferrin exhibited an increased signal-to-noise ratio compared to that of direct ESI-MS analysis due to online purification of the protein sample and size separation of dimers with AF4.

Effect of asymmetrical flow field-flow fractionation channel geometry on separation efficiency

The separation efficiencies of three different asymmetrical flow field-flow fractionation (AF4) channel designs were evaluated using polystyrene latex standards. Channel breadth was held constant for one channel (rectangular profile), and was reduced either linearly (trapezoidal profile) or exponentially (exponential profile) along the length for the other two. The effective void volumes of the three channel types were designed to be equivalent. Theoretically, under certain flow conditions, the mean channel flow velocity of the exponential channel could be arranged to remain constant along the channel length, thereby improving separation in AF4. Particle separation obtained with the exponential channel was compared with particle separation obtained with the trapezoidal and rectangular channels. We demonstrated that at a certain flow rate condition (outflow/inflow rate = 0.2), the exponential channel design indeed provided better performance with respect to the separation of polystyrene nanoparticles in terms of reducing band broadening. While the trapezoidal channel exhibited a little poorer performance than the exponential, the strongly decreasing mean flow velocity in the rectangular channel resulted in serious band broadening, a delay in retention time, and even failure of larger particles to elute.

New methods allowing the detection of protein aggregates

Aggregation compromises the safety and efficacy of therapeutic proteins. According to the manufacturer, the therapeutic immunoglobulin trastuzumab (Herceptin®) should be diluted in 0.9% sodium chloride before administration. Dilution in 5% dextrose solutions is prohibited. The reason for the interdiction is not mentioned in the Food and Drug Administration (FDA) documentation, but the European Medicines Agency (EMEA) Summary of Product Characteristics states that dilution of trastuzumab in dextrose solutions results in protein aggregation. In this paper, asymmetrical flow field-flow fractionation (FFF), fluorescence spectroscopy, fluorescence microscopy and transmission electron microscopy (TEM) have been used to characterize trastuzumab samples diluted in 0.9% sodium chloride, a stable infusion solution, as well as in 5% dextrose (a solution prone to aggregation). When trastuzumab samples were injected in the FFF channel using a standard separation method, no difference could be seen between trastuzumab diluted in sodium chloride and trastuzumab diluted in dextrose. However, during FFF measurements made with appropriate protocols, aggregates were detected in 5% dextrose. The parameters enabling the detection of reversible trastuzumab aggregates are described. Aggregates could also be documented by fluorescence microscopy and TEM. Fluorescence spectroscopy data were indicative of conformational changes consistent with increased aggregation and adsorption to surfaces. The analytical methods presented in this study were able to detect and characterize trastuzumab aggregates.

Molecular weight and structure characterization of sodium hyaluronate and its gamma radiation degradation products by flow field-flow fractionation and on-line multiangle light scattering

A combined flow field-flow fractionation (FlFFF)/multiangle light scattering (MALS)/differential refractive index (DRI) detection method has been utilized for the size fractionation and characterization of ultrahigh molecular weight (MW) sodium hyaluronate (NaHA) samples. Separation of broad MW NaHA polymers was carried out by a frit inlet asymmetrical FlFFF channel employed with a linear field programming method followed by the on-line monitoring of light scattering at multiple angles for the calculation of MW and for the study of the conformation of NaHA samples. NaHA samples examined were: (1) two different viscosity fractions of NaHA obtained by a refinement process and (2) NaHA products of gamma radiation degradation. While the NaHA samples of two different viscosity fractions exhibited clearly different MW distributions and similar conformation, the radiation degraded NaHA samples showed a clear difference in both MW distribution and polymer structure.

Separation and Characterization of Particles in Natural Water and Soil Using Flow Field-Flow Fractionation

Flow field-flow fractionation (FlFFF) is used to characterize particles in natural water (ground and surface water) and soil. The opposed flow sample concentration (OFSC) mode of FlFFF (OFSC-FlFFF) is employed, where the colloidal sample is continuously fed into the channel so that the particles are focused into a narrow band near the inlet of the FlFFF channel before the separation is initiated. There is no need for stopping the flow for the sample relaxation, which is usually required in conventional FlFFF operations. First, the OFSC-FlFFF is tested with mixtures of polystyrene latex spheres. Then the OFSC-FlFFF procedure is optimized for the analysis of particles in natural water and soil by varying various experimental parameters including the flow rates. Ground water of up to 100 mL has been successfully loaded, concentrated, and characterized by OFSC-FlFFF. Results show that the OFSC-FlFFF provides a simplified alternative to existing off-line concentration procedures, and it shows high potential for application to analysis of dilute colloidal particles in environmental water. The composition of the samples was analyzed using atomic absorption spectrometry.

Characterization of functionalized styrene–butadiene rubber by flow field-flow fractionation/light scattering in organic solvent

Flow field-flow fractionation (FlFFF) using an organic solvent as mobile phase has been effectively utilized for the separation and characterization of functionalized styrene–butadiene rubbers (SBR) that are polymerized and followed by coupling reaction in solution. Separation of broad molecular weight SBR was accomplished by an asymmetrical FlFFF channel in THF under field programming and the molecular weight distribution (MWD) of the SBR sample was determined by on-line measurement of light scattering. In this study, FlFFF has been utilized to characterize high-MW functionalized SBR from the low-MW non-functionalized molecules which were used for coupling reaction to produce high-MW functionalized SBRs, and to determine the coupling number of the functionalized SBRs depending on the type of the coupling reagents. The resulting MWD of the SBR samples prepared by the different coupling reagents (SnCl 4 and a polydimethylsiloxane compound) were compared.

Matrix removal before inductively coupled plasma spectrometric detection: another capability of flow field-flow fractionation

A modified flow field-flow fractionation (FlFFF) configuration was investigated for its matrix removal capability before inductively coupled plasma optical emission spectrometric (ICP-OES) detection. Poly(ethylene imine) (PEI) having a molecular weight of 25000 Da was added to test solutions (buffered at pH 9) to form complexes with analyte elements (Ag, Cd, Co, Cu, Hg, Ni, Pb and Zn) but not with the matrix elements (Ca, K, Mg and Na). With the use of a poly(ether sulfone) membrane having a 1000 Da molecular weight cut-off inside the FlFFF channel, matrix elements were removed by filtering off through the membrane, whereas the PEI–analyte complexes remained in the channel and were subsequently eluted and detected. Two approaches, i.e., cross flow matrix removal (CFM) and opposing flow matrix removal-analyte preconcentration (OFM), were evaluated for matrix removal and matrix removal with simultaneous preconcentration, respectively. With the FlFFF/CFM approach, the signal intensities of matrix elements were reduced by approximately 80–90% while those of analyte elements were only slightly reduced (∼20%). With the FlFFF/OFM approach, an unlimited sample volume can be introduced into the FlFFF channel with an elution volume of approximately 1 ml, leading to analyte preconcentration.

Separation of Proteins Mixture in Hollow Fiber Flow Field-Flow Fractionation

Jin-Won Park, Ik-Sung An, and Kangtaek LeeDepartment of Chemical Engineering, Yonsei University, Seoul 120-749, KoreaReceived May 28, 2003Flow field-flow fractionation (FlFFF) is a technology to separate the molecules by size in an open channel.Molecules with different size have different diffusivities and are located vertically in different positions whenpassing through an open channel. In this study, hollow fiber membranes instead of conventional rectangularchannels have been used as materials for the open channel and this change would decrease the cost ofmanufacturing. FlFFF is a useful technique to characterize the biopolymeric materials. Retention time,diffusion coefficients and Stokes radius of analysis can be calculated from the related simple equations.Hollow-fiber flow field-flow fractionation (HF-FlFFF) has been used for the characterization and separation ofprotein mixture in a phosphate buffer solution and has demonstrated the potential to be developed into adisposable FlFFF channel. The important indexes for the analytical separation are selectivity, resolution andplate height. The optimized separation condition for protein mixture of Ovalbumin, Alcohol dehydrogenase,Apoferritin and Thyroglobulin is = 0.65/0.85 mL/min. Key Words : Hollow fiber flow FFF, Protein mixture, Retention timeIntroductionHollow fiber flow FFF (HF-FlFFF) is a new separationmethod for polymers and particles. As opposed to thenormal arrangement with two parallel plates as a separationchannel in Flow Field-Flow Fractionation (FlFFF), cylindri-cal hollow fiber FFF has certain advantages, such assimplicity of operation and independent control of the twoflows (elution and cross flow), including an easier way ofperforming gradient elution. The cylindrical channelautomatically maintains its cylindrical cross section whenpressurized.

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.

Frit inlet/frit outlet flow field-flow fractionation: methodology for colored dissolved organic material in natural waters

Results from the first application FIFO–FlFFF (1 frit inlet/frit outlet–flow field-flow fractionation) to the characterization of colored dissolved organic material (CDOM) in river and coastal waters are described. FlFFF separates compounds based on their diffusional characteristics. The frit inlet/frit outlet (FIFO) feature was recently developed to concentrate samples inside the FlFFF channel for improved stability, reproducibility and sensitivity. Our results show that FIFO increases sensitivity by an order of magnitude and the system has a linear response to molecular weight and concentration over a wide dynamic range. Errors for replicate analyses with a Caloosahatchee River sample from southwest Florida were <2.5% for retention time and <4% for peak areas, indicating good reproducibility and precision for the analysis of CDOM in natural water samples. However, recovery rates were low (35–65% for fresh waters and 5.7–8.1% for marine waters) and it will be necessary to test different membranes and lower molecular weight cut-off to improve recoveries in future studies. Molecular weights were calculated from the measured retention times and log-linear calibration curves generated with polystyrene sulfonate standards (1800–35,000 Da). In river water, ∼98% of CDOM occurred in a fraction centered around 2 kDa and ∼2% in a fraction with a maximum at 13 kDa (based on maximum peak intensity for absorbance at 330 nm and fluorescence at 350–450 nm). This minor higher molecular weight fraction was completely gone in offshore coastal waters. This loss may be due to dilution of the river samples and/or biological and photochemical degradation of CDOM. Irradiating a river sample with sunlight produced a fractogram characteristic of marine waters, suggesting that photodegradation is an important mechanism in altering the molecular and optical characteristics of CDOM.

Modeling of sample dynamics in rectangular asymmetrical flow field-flow fractionation channels

We model the evolution of the concentration field of macromolecules in a rectangular asymmetrical flow field-flow fractionation channel using center manifold techniques. The deviation of the primary flow from a parabolic profile influences the concentration field and this is investigated to complement previously known results. The long-term evolution of the components of the sample is shown to be well described by a one-dimensional advection-diffusion equation. The coefficients of this equation are determined by the rigorous analysis of the complete set of equations governing the two-dimensional fluid flow. This model gives quantitative predictions of the elution time of the samples, the width of the concentration pulse, and the resolution of the apparatus. The influence of initial sample width and effects of the secondary relaxation from focusing to elution conditions are discussed. Reported theoretical predictions are in agreement with experimental results published previously.

Newly designed flow field-flow fractionation channel for macromolecules and particles in the submicrometer and micrometer range

The flow field-flow fractionation (FIFFF) technique is a promising method for separating and analysing particles and large size macromolecules from a few nanometers to approximately 50 μm. A new fractionation channel is described featuring well defined flow conditions even for low channel heights with convenient assembling and operations features. The application of the new flow field-flow fractionation channel is proved by the analysis of pigments and other small particles of technical interest in the submicrometer range. The experimental results including multimodal size distributions are presented and discussed.

Design of an asymmetrical flow field-flow fractionation channel for uniform channel flow velocity

A new design for an asymmetrical flow field-flow fractionation (FFF) channel, having a breadth that decreases exponentially along its length, is presented. It is shown that such a channel may be operated under certain flow conditions to obtain a constant mean channel flow velocity throughout its effective length. This is not possible with trapezoidal channels, which were themselves introduced with the objective of reducing the variation of mean flow velocities along the channel length. It is also shown that when operated under other flow conditions, the resulting variation in mean flow velocity is reduced in comparison to that predicted for trapezoidal channels. © 1997 John Wiley & Sons, Inc. J Micro Sep9: 459–467, 1997

Flow field-flow fractionation in the study of dairy products

The performance of flow field-flow fractionation (FlFFF) was studied in the particle size analysis of dairy products including whey proteins, casein micelles, and fat globules, over a wide size range. The products investigated were full-fat milk, skim milk, cream, ice cream, powdered milk, nonlactose milk, and infant formula. In addition, three bacteria (Pediococcus acidilactici streptococcus, Propionibacterium, and Lactococcus lactic micrococcus) related to dairy processing were examined. FlFFF results were found to be consistent with literature values and data obtained by dynamic light scattering. A very-low-ionic-strength carrier had to be used to avoid complete adsorption of the casein micelles on the membrane inside the FlFFF channel. The observed particle size was also found to depend on the ionic strength and flow rate of the carrier. These dependencies were attributed to the electrostatic interaction between casein micelles and the channel wall and variations an the micelle size with ionic strength. Of the three membranes tested, regenerated cellulose and polyolefin materials produced similar particle size values, whereas the values obtained with polysulfone membrane were too high. When the pH of the carrier was raised in FlFFF, the casein peak shifted to a lower particle size, but when the pH was increased in light-scattering studies, a constant size was obtained. © 1997 John Wiley & Sons, Inc. J Micro Sep9: 601–609, 1997

High-speed particle separation and steric inversion in thin flow field-flow fractionation channels

The performance of thin (down to 71 μm thick) flow field-flow fractionation (flow FFF) channels was examined at both high channel flow-rates (up to ∼47 ml/min) and cross flow-rates (up to ∼11 ml/min). High levels of retention were observed, suggestive of good performance characteristics. Supporting this expectation, four sizes (0.04–0.30 μm) of polysterene latex microbeads were baseline separated in under 3 min. Nonetheless, a plate height analysis showed that performance was still less than theoretically expected. Fast steric-hyperlayer separations of larger latex beads were observed in the same systems. Furthermore, it was shown that the steric inversion diameter was shifted down to ∼0.23 μm, thus expanding the size range to which this FFF mode is applicable. The steric inversion phenomenon observed using narrow latex standards was shown to be consistent with that found for a polydisperse polyvinylchloride latex.

Separation speed, retention, and dispersion in asymmetrical flow field-flow fractionation as functions of channel dimensions and flow rates

The theory for the asymmetrical flow field-flow fractionation channel was evaluated experimentally. The assumption of a constant crossflow along the channel was satisfactory. The zone-broadening theory was extended to take into a account the longitudinal diffusion and the width of the starting sample zone. In the present apparatus the longitudinal diffusion was negligible but may have to be considered for lower outlet flow rates. The axial flow velocity gradient has the advantage that the contribution by the width of the starting sample zone to the final zone width decreases

Separation and molecular weight distribution of anionic and cationic water-soluble polymers by flow field-flow fractionation

A broad study is reported here on the retention and fration of anionic polystyrenesulfonate (PSS) and cationic polyvinylpyridine (PVP) water-soluble polymers by flow field-flow fractionation (FFF) using a thin hydrophobic polypropylene membrane as the accumulation wall of the FFF channel. As background, the general role and impact of polymer adsorption in size exclusion chromatography and in flow FFF are compared. Adsorption at the accumulation wall in FFF systems is described, and explanations for its self-limitation and nonobtrusion are supported by experimental data

Zone broadening and dilution in rectangular and trapezoidal asymmetrical flow field-flow fractionation channels

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTZone broadening and dilution in rectangular and trapezoidal asymmetrical flow field-flow fractionation channelsAnne. Litzen and Karl Gustav. WahlundCite this: Anal. Chem. 1991, 63, 10, 1001–1007Publication Date (Print):May 15, 1991Publication History Published online1 May 2002Published inissue 15 May 1991https://doi.org/10.1021/ac00010a013RIGHTS & PERMISSIONSArticle Views497Altmetric-Citations138LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (825 KB) Get e-Alerts Get e-Alerts