Field-flow Fractionation System Research Articles

The Development of an AF4 Characterization Pipeline for Polymeric Nanoparicle-Protein Interactions

The centrifugation-wash method is currently the standard technique used for the recovery of nanoparticle-protein complexes from protein-containing serum. However, this technique is disruptive, and leads to changes in nanoparticle parameters and the surface-bound protein composition. In this study, we explore the use of field flow fractionation (FFF) for the development of a robust pipeline for the characterization of nanoparticle parameters following protein corona formation under physiologically-relevant conditions. Model surface-modified polystyrene latex nanoparticles were treated with protein-containing medium mimicking cell-culture conditions under physiologically-relevant conditions. Following incubation, nanoparticle-protein samples were then fractionated using the FFF system and characterized using in-line detectors. Conventional nanoparticle-protein studies typically use the highly invasive centrifugation-wash method which leads to changes in measured particle parameters. Our studies show that AF4 allows for high resolution and characterization of in-situ nanoparticle-protein complexes. This work will allow for the development of a more harmonized approach for characterizing nanoparticle-protein interactions under biologically-relevant conditions.

Revealing cholesterol effects on PEGylated HSPC liposomes using AF4-MALS and simultaneous small- and wide-angle X-ray scattering.

Liposome development is of great interest owing to increasing requirements for efficient drug carriers. The structural features and thermal stability of such liposomes are crucial in drug transport and delivery. Reported here are the results of the structural characterization of PEGylated liposomes via small- and wide-angle X-ray scattering and an asymmetric flow field-flow fractionation (AF4) system coupled with differential refractive-index detection, multi-angle light scattering (MALS) and dynamic light scattering. This integrated analysis of the exemplar PEGylated liposome formed from hydrogenated soy phosphatid-yl-choline (HSPC) with the addition of cholesterol reveals an average hydro-dynamic radius (R h) of 52 nm with 10% polydispersity, a comparable radius of gyration (R g) and a major liposome particle mass of 118 kDa. The local bilayer structure of the liposome is found to have asymmetric electronic density profiles in the inner and outer leaflets, sandwiched by two PEGylated outer layers ca 5 nm thick. Cholesterol was found to effectively intervene in lipid chain packing, resulting in the thickening of the liposome bilayer, an increase in the area per lipid and an increase in liposome size, especially in the fluid phase of the liposome. These cholesterol effects show signs of saturation at cholesterol concentrations above ca 1:5 cholesterol:lipid molar ratio.

Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor

Continuous flow quartz crystal microbalance (QCM) was utilized to study binding kinetics between EV subpopulations (exomere- and exosome-sized EVs) and four affinity ligands: monoclonal antibodies against tetraspanins (anti-CD9, anti-CD63, and anti-CD81) and recombinant intercellular adhesion molecule-1 (ICAM-1) or CD54 protein). High purity CD9+, CD63+, and CD81+ EV subpopulations of <50 nm exomeres and 50–80 nm exosomes were isolated and fractionated using our recently developed on-line coupled immunoaffinity chromatography – asymmetric flow field-flow fractionation system. Adaptive Interaction Distribution Algorithm (AIDA), specifically designed for the analysis of complex biological interactions, was used with a four-step procedure for reliable estimation of the degree of heterogeneity in rate constant distributions. Interactions between exomere-sized EVs and anti-tetraspanin antibodies demonstrated two interaction sites with comparable binding kinetics and estimated dissociation constants Kd ranging from nM to fM. Exomeres exhibited slightly higher affinity compared to exosomes. The highest affinity with anti-tetraspanin antibodies was achieved with CD63+ EVs. The interaction of EV subpopulations with ICAM-1 involved in cell internalization of EVs was also investigated. EV – ICAM-1 interaction was also of high affinity (nM to pM range) with overall lower affinity compared to the interactions of anti-tetraspanin antibodies and EVs. Our findings proved that QCM is a valuable label-free tool for kinetic studies with limited sample concentration, and that advanced algorithms, such as AIDA, are crucial for proper determination of kinetic heterogeneity. To the best of our knowledge, this is the first kinetic study on the interaction between plasma-derived EV subpopulations and anti-tetraspanin antibodies and ICAM-1.

Development of an asymmetrical flow field-flow fractionation system for the size characterization of starch granules

淀粉颗粒粒径与分子尺寸分别在1~100 μm和20~250 nm之间,是影响淀粉功能特性的重要因素之一。非对称场流分离(AF4)是一种基于样品与外力场相互作用机制的分离技术,已应用于表征淀粉分子尺寸分布。商品化的AF4系统的粒径检测范围为1 nm~10 μm,对于淀粉颗粒粒径表征具有一定的局限性。该文研制了AF4分离系统;考察了其在微米尺度下对红薯、莲子和大米淀粉颗粒粒径表征的性能;采用微米尺寸的聚苯乙烯乳化球(PS)标准样品验证了构建的AF4系统的分离性能。实验结果显示,构建的AF4系统对PS混合样品(粒径2、6、12、20 μm)实现了基线分离,同商品化AF4相比提高了检测上线,具有分离表征淀粉颗粒的潜力。此外,该文研究了载液组成对淀粉颗粒分离表征的影响;通过光学显微镜验证了构建的AF4系统在微米尺度上对淀粉颗粒粒径分布的表征能力。最后,采用商品化的AF4系统串联多角度激光光散射检测器和示差折光检测器对3种淀粉分子进行了分离表征,考察了淀粉的溶解温度对其表征结果的影响。在摩尔质量10 6~108 g/mol范围内,红薯和莲子淀粉的回转半径和水合半径的比值(Rg/Rh)在0.9~1.1之间,大米淀粉的Rg/Rh在1.2~1.4之间。实验结果证明构建的AF4系统是一种快速、准确的淀粉颗粒粒径表征方法,与商品化的AF4系统结合可为研究淀粉尺寸分布与其功能性质之间的关系提供技术支持。

Optimization of hyphenated asymmetric flow field-flow fractionation for the analysis of silver nanoparticles in aqueous solutions

The extensive use of silver nanoparticles (AgNPs) in consumer products, medicine, and industry leads to their release into the environment. Thus, a characterization of the concentration, size, fate, and toxicity of AgNPs under environmental conditions is required. In this study, we present the characterization and optimization of an asymmetric flow field-flow fractionation (AF4) system coupled with UV/Vis spectrophotometer and dynamic light scattering (DLS) detector as a powerful tool for the size separation and multi-parameter characterization of AgNPs in complex matrices. The hyphenated AF4-UV/Vis-DLS system was first characterized using individual injections of the different size fractions. We used electrostatically stabilized AgNPs of 20-, 50-, and 80-nm nominal diameters coated with lipoic acid. We investigated the effect of applied cross-flows, carrier solutions, focus times, and quantity of injected particles on the nature of the AF4 fractograms and on the integrity of the AgNPs. Best size separation of a 1:1 mixture of 20- and 80-nm AgNPs was achieved using cross-flows of 0.5 and 0.7 mL/min with 1 mM NaCl and 0.05% v/v Mucasol as carrier solutions. We also researched the behavior of AgNPs in natural waters using the hyphenated AF4-UV/Vis-DLS system, under determined optimal conditions.Graphical abstractSchematic and photograph of the AF4 setup with numbered hardware devices. Dashed lines represent electrical connections; continuous lines represent fluidic connections. For a better overview, not all fluidic connections between pump/6-way valve (2) and the Eclipse AF4 device (3) are shown in the schematic. The fluorescence detector (FL (7)) was not used in the study presented herein.

Continuous asymmetrical flow field-flow fractionation for the purification of proteins and nanoparticles

We present a novel continuous two-dimensional asymmetrical flow field-flow fractionation (2D-AF4) system that is able to fractionate a feed solution of nano-sized solutes (e.g., proteins, nanoparticles) according to their size in aqueous solvents. The key component that generates the continuous separation is a microstructured ultrafiltration membrane with slanted grooves on its surface. The solutes are migrating over the grooves, which are causing a lateral displacement from the direction of the main channel flow, and they are exiting the channel at different outlets. The deflection angle depends on the mean layer thickness of the solutes and consequently on their hydrodynamic radius; with a specific cross-flow, larger solutes exhibit a larger deflection angle which results in a spatial separation. By adjusting the outlet flow and the cross-flow rate, the system can be optimized with respect to purity, recovery and speed. A prototype device has been designed and tested. A proof of principle of the continuous fractionation is demonstrated with mixtures of two model proteins, apoferritin (443 kDa) and thyroglobulin (669 kDa), and of two polystyrene latex standards with diameters of 34 and 102 nm.

Application of microstructured membranes for increasing retention, selectivity and resolution in asymmetrical flow field-flow fractionation

In the present proof-of-concept study, we demonstrate that retention time, selectivity and resolution can be increased in asymmetrical flow field-flow fractionation (AF4) by introducing microstructured ultrafiltration membranes. Evenly spaced micron-sized grooves, that are placed perpendicular to the channel flow on the accumulation wall of a field-flow fractionation system, cause a decrease in the zone velocity which is stronger for larger solutes. This has been demonstrated in thermal field-flow fractionation, and we prove that this is also the case in AF4. We examine the hypothesis theoretically and experimentally, by both computational and physical experiments. By means of moment analysis, we derive theoretically a set of equations which, under certain conditions, describe the mass transport and relate retention time, selectivity and plate height to the dimensions of the grooves. Physical experiments are carried out using microstructured polyethersulfone membranes fabricated by hot embossing, and the experimental results are compared with computational fluid dynamics experiments.

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.

Application of asymmetric flow field-flow fractionation (AF4) and multiangle light scattering (MALS) for the evaluation of changes in the product molar mass during PVP-b-PAMPS synthesis

The use of polymers for the delivery of drugs has increased dramatically in the last decade. To ensure the desired properties and functionality of such substances, adequate characterization in terms of the molar mass (M) and size is essential. The aim of this study was to evaluate the changes in the M and size of PVP-b-PAMPS when the amounts of the synthesis reactants in the two-step radical reaction were varied. The determination of the M and size distributions was performed by an asymmetric flow field-flow fractionation (AF4) system connected to multiangle light scattering (MALS) and differential refractive index (dRI) detectors. The results show that the M of the polymers varies depending on the relative amounts of the reactants and that AF4-MALS-dRI is a powerful characterization technique for analyzing polymers. Using AF4, it was possible to separate the product of the first radical reaction (PVP-CTA) into two populations. The first population had an elongated, rod-like or random coil conformation, and the second had a conformation corresponding to homogeneous spheres or a microgel structure. PVP-b-PAMPS had only one population, which had a rod-like conformation. The molar masses of PVP-CTA and PVP-b-PAMPS found in this study were higher than those reported in previous studies.

Determination of length distribution of TEMPO-oxidized cellulose nanofibrils by field-flow fractionation/multi-angle laser-light scattering analysis

Aqueous nanocellulose dispersions were prepared from wood cellulose by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation. The obtained TEMPO-oxidized cellulose was converted into TEMPO-oxidized cellulose nanofibrils (TOCNs) of different lengths by controlling the nanofibrillation conditions in water or using dilute acid hydrolysis. The average lengths and length distributions of TOCNs have been measured from transmission electron microscopy (TEM) and atomic force microscopy (AFM) images. However, because the number of nanocelluloses observable in TEM and AFM images is limited, a more reliable method is needed to obtain the lengths/length distributions of TOCNs. In this study, the aqueous TOCN dispersions were subjected to a combination of field-flow fractionation (FFF) and multi-angle laser-light scattering (MALLS). The optimum FFF operation conditions for the acid-hydrolyzed TOCN were first established to obtain reasonable data. For TOCNs with average lengths > 400 nm, suitable separation could not be achieved using the FFF/MALLS system. In contrast, the TOCNs with average lengths of 170 and 270 nm were adequately separated according to their sizes by the FFF system. The TOCN length distribution patterns corresponded well to those obtained from TEM images. However, the amounts of TOCNs with lengths > 250 nm were underestimated compared with those determined from TEM images. For TOCNs with average lengths of 170 and 270 nm, the molar mass at each TOCN length was determined using the FFF/MALLS system combined with a refractive index detector, where a specific refractive index increment of 0.165 mL/g was used for TOCN.

Characterization of Ultrahigh-Molecular-Weight Oilfield Polyacrylamides Under Different pH Environments by Use of Asymmetrical-Flow Field-Flow Fractionation and Multiangle-Light-Scattering Detector

Summary Various types of ultrahigh-molar-mass polyacrylamides (HPAMs) and their copolymers and terpolymers used not only in enhanced oil recovery (EOR) but also in drilling, fracturing, water treatment, and tailing applications require an accurate description of polymer molar mass (Mw) and hydrodynamic size for their optimal design. The range of Mw for various types of available HPAMs is between 4 and 30 million g/mol and is typically determined by use of intrinsic-viscosity measurement. Molecular-weight distribution (MWD) cannot be determined because neither standard with low polydispersity index (PDI) nor gel-permeation-chromatography (GPC) or size-exclusion-chromatography (SEC) techniques exist today for such ultrahigh-molar-mass polymers. Moreover, the solution environment in underground reservoirs, characterized by high temperatures, pH values, and the presence of monovalent and divalent ions, may often lead to changes in polymer-macromolecular conformation. Current techniques, SEC, ultraviolet-visible measurements, and liquid chromatography, are not capable of accurately investigating these complex macromolecular structures for various reasons. In this paper, the asymmetrical-flow field-flow fractionation (AF4) system was used to fractionate four different ultrahigh-molecular-weight HPAM samples, varying in molar mass and commercially used for oilfield applications, in various carrier pH values ranging from 12 to 3 (pH values of 12, 7.4, and 3). The system uses field-flow fractionation (FFF), a family of analytical techniques developed specifically for separating and characterizing macromolecules, colloids, and particles. The theoretical separation range for AF4 is between 103 to 1012 g/mol. Other advantages over conventional GPC/SEC include minimum shear degradation, mild operating conditions, and no sample loss caused by adsorption. The flow system was equipped with a multiangle-light-scattering (MALS) and refractive-index (RI) detectors to measure molar mass and radius of gyration (Rg). The results show that the observed molecular weight of the polymer aggregate increased substantially as the pH value of the carrier solution decreased from 12 to 3, especially for higher-molar-mass polymers. The sample Rg showed the opposite trend, decreasing as the pH of the carrier solution changed from basic to acidic. For ultrahigh molecular HPAM at high pH, a narrower molar mass and radius distribution was observed with disaggregated molar mass and increased branching or swelling (therefore larger hydrodynamic radius). Use of this direct separation and measurement technique can improve understanding of polymer-macromolecular structure and corresponding changes in the reservoir brines.

Particle Based Modeling of Electrical Field Flow Fractionation Systems

Electrical Field Flow Fractionation (ElFFF) is a sub method in the field flow fractionation (FFF) family that relies on an applied voltage on the channel walls to effect a separation. ElFFF has fallen behind some of the other FFF methods because of the optimization complexity of its experimental parameters. To enable better optimization, a particle based model of the ElFFF systems has been developed and is presented in this work that allows the optimization of the main separation parameters, such as electric field magnitude, frequency, duty cycle, offset, flow rate and channel dimensions. The developed code allows visualization of individual particles inside the separation channel, generation of realistic fractograms, and observation of the effects of the various parameters on the behavior of the particle cloud. ElFFF fractograms have been generated via simulations and compared with experiments for both normal and cyclical ElFFF. The particle visualizations have been used to verify that high duty cycle voltages are essential to achieve long retention times and high resolution separations. Furthermore, by simulating the particle motions at the channel outlet, it has been demonstrated that the top channel wall should be selected as the accumulation wall for cyclical ElFFF to reduce band broadening and achieve high efficiency separations. While the generated particle based model is a powerful tool to estimate the outcomes of the ElFFF experiments and visualize particle motions, it can also be used to design systems with new geometries which may lead to the design of higher efficiency ElFFF systems. Furthermore, this model can be extended to other FFF techniques by replacing the electrical field component of the model with the fields used in the other FFF techniques.

A critical evaluation of an asymmetrical flow field-flow fractionation system for colloidal size characterization of natural organic matter

Colloidal retention characteristics, recovery and size distribution of model macromolecules and natural dissolved organic matter (DOM) were systematically examined using an asymmetrical flow field-flow fractionation (AFlFFF) system under various membrane size cutoffs and carrier solutions. Polystyrene sulfonate (PSS) standards with known molecular weights (MW) were used to determine their permeation and recovery rates by membranes with different nominal MW cutoffs (NMWCO) within the AFlFFF system. Based on a ≥90% recovery rate for PSS standards by the AFlFFF system, the actual NMWCOs were determined to be 1.9kDa for the 0.3kDa membrane, 2.7kDa for the 1kDa membrane, and 33kDa for the 10kDa membrane, respectively. After membrane calibration, natural DOM samples were analyzed with the AFlFFF system to determine their colloidal size distribution and the influence from membrane NMWCOs and carrier solutions. Size partitioning of DOM samples showed a predominant colloidal size fraction in the <5nm or <10kDa size range, consistent with the size characteristics of humic substances as the main terrestrial DOM component. Recovery of DOM by the AFlFFF system, as determined by UV-absorbance at 254nm, decreased significantly with increasing membrane NMWCO, from 45% by the 0.3kDa membrane to 2–3% by the 10kDa membrane. Since natural DOM is mostly composed of lower MW substances (<10kDa) and the actual membrane cutoffs are normally larger than their manufacturer ratings, a 0.3kDa membrane (with an actual NMWCO of 1.9kDa) is highly recommended for colloidal size characterization of natural DOM. Among the three carrier solutions, borate buffer seemed to provide the highest recovery and optimal separation of DOM. Rigorous calibration with macromolecular standards and optimization of system conditions are a prerequisite for quantifying colloidal size distribution using the flow field-flow fractionation technique. In addition, the coupling of AFlFFF with fluorescence EEMs could provide new insights into DOM heterogeneity in different colloidal size fractions.

Size and concentration determination of (functionalised) fullerenes in surface and sewage water matrices using field flow fractionation coupled to an online accurate mass spectrometer: Method development and validation

In order to assess the environmental risks of a compound it is imperative to have suitable and reliable techniques for its determination in environmental matrices. In this paper, we focused on a method development for the recently introduced online coupling of a field flow fractionation (FFF) system to an Orbitrap-HRMS, that allows the simultaneous size and concentration determination of different aqueous fullerene aggregates and their concentrations in different size fractions. A 0.05% NH4OH solution in water was identified as the best carrier liquid for the analysis of the three different aqueous fullerene suspensions (C60 [60], [6,6]-phenyl-C61 butyric acid methyl ester ([60]PCBM) and [6,6]-(bis)phenyl-C61 butyric acid methyl ester ([60]bisPCBM)). The multi-angle light scattering (MALS) data received after employing the ammonia solution was consistent with both the theory and calibration using well defined Au and latex particles. The LODs obtained using Orbitrap HRMS detection were 0.1μgL−1 for an injection volume of 100μL which are significantly better than the LODs obtained by using UV (20μgL−1) and MALS detectors (5μgL−1). However, these LODs can be further improved as in theory there is no limit to the amount of sample that can be injected into the FFF. Environmental samples (river and sewage water) were spiked with fullerenes and the fractograms obtained for these samples revealed that the matrix does affect the size of fullerene aggregates. Information on the size distribution can be useful for the risk assessment of these particles.

Circuit modification in electrical field flow fractionation systems generating higher resolution separation of nanoparticles

Compared to other sub-techniques of field flow fractionation (FFF), cyclical electrical field flow fractionation (CyElFFF) is a relatively new method with many opportunities remaining for improvement. One of the most important limitations of this method is the separation of particles smaller than 100nm. For such small particles, the diffusion rate becomes very high, resulting in severe reductions in the CyElFFF separation efficiency. To address this limitation, we modified the electrical circuitry of the ElFFF system. In all earlier ElFFF reports, electrical power sources have been directly connected to the ElFFF channel electrodes, and no alteration has been made in the electrical circuitry of the system. In this work, by using discrete electrical components, such as resistors and diodes, we improved the effective electric field in the system to allow high resolution separations. By modifying the electrical circuitry of the ElFFF system, high resolution separations of 15 and 40nm gold nanoparticles were achieved. The effects of applying different frequencies, amplitudes and voltage shapes have been investigated and analyzed through experiments.

Separation of nano- and micro-sized materials by hyphenated flow and centrifugal field-flow fractionation

A novel field-flow fractionation (FFF) separation system, a separation system that hyphenated a flow FFF and a centrifugal FFF, was investigated as a hybrid tool for fractionation of nano- and micro-materials. The investigated FFF system makes it possible to separate materials with a considerably wider size distribution than is possible by either a flow FFF or centrifugal FFF alone. The hyphenated FFF separation system is also able to simultaneously separate materials by both hydrodynamic size and density. This investigated analytical method should play an important role in developing new applications of colloidal nano- and micro-materials in industrial and biological research.

Application of asymmetrical flow field-flow fractionation for size characterization of low density lipoprotein in egg yolk plasma

Home-made asymmetrical flow field-flow fractionation (AF4) system, online coupled with ultraviolet/visible (UV/Vis) detector was employed for the separation and size characterization of low density lipoprotein (LDL) in egg yolk plasma. At close to natural condition of egg yolk, the effects of cross flow rate, sample loading, and type of membrane on the size distribution of LDL were investigated. Under the optimal operation conditions, AF4-UV/Vis provides the size distribution of LDL. Moreover, the precision of AF4-UV/Vis method proposed in this work for the analysis of LDL in egg yolk plasma was evaluated. The intra-day precisions were 1.3% and 1.9% (n=7) and the inter-day precisions were 2.4% and 2.3% (n=7) for the elution peak height and elution peak area of LDL, respectively. Results reveal that AF4-UV/Vis is a useful tool for the separation and size characterization of LDL in egg yolk plasma.

Cyclical magnetic field flow fractionation

In this study, a new magnetic field flow fractionation (FFF) system was designed and modeled by using finite element simulations. Other than current magnetic FFF systems, which use static magnetic fields, our system uses cyclical magnetic fields. Results of the simulations show that our cyclical magnetic FFF system can be used effectively for the separation of magnetic nanoparticles. Cyclical magnetic FFF system is composed of a microfluidic channel (length = 5 cm, height = 30 μm) and 2 coils. Square wave currents of 1 Hz (with 90 deg of phase difference) were applied to the coils. By using Comsol Multiphysics 3.5a, magnetic field profile and corresponding magnetic force exerted on the magnetite nanoparticles were calculated. The magnetic force data were exported from Comsol to Matlab. In Matlab, a parabolic flow profile with maximum flow speed of 0.4 mL/h was defined. Particle trajectories were obtained by the calculation of the particle speeds resulted from both magnetic and hydrodynamic forces. Particle trajectories of the particles with sizes ranging from 10 to 50 nm were simulated and elution times of the particles were calculated. Results show that there is a significant difference between the elution times of the particles so that baseline separation of the particles can be obtained. In this work, it is shown that by the application of cyclical magnetic fields, the separation of magnetic nanoparticles can be done efficiently.

Optimization and characterization of a microscale thermal field-flow fractionation system

A thorough investigation of the design considerations for microscale thermal field-flow fractionation and characterization of a 25μm thin microscale thermal field-flow fractionation system is reported. A 4–50 times volume reduction from mesoscale and macroscale systems warrants customized design and operational conditions for microscale separation systems. Theoretical calculations are done to illustrate the importance of the increased dispersion due to extra-column tubing, off-chip detection and sample injection volume with reduced channel dimensions. An optimized microscale thermal field-flow fractionation (ThFFF) channel is fabricated using rapid and cost effective manufacturing and assembly processes. Specifically, improvements in material selection and arrangement are implemented to achieve higher particle retentions. The new instrument arrangement includes high conductivity silicon as the cold wall and a thin polymer layer with low thermal conductivity as the hot wall which results in high temperature gradients (∼106°C/m) across the microchannel and subsequently high retention. Single particle retention separations are carried out with polystyrene nanoparticle samples in an aqueous carrier to characterize the device and demonstrate the improvements.

Characterization of a microscale thermal–electrical field-flow fractionation system

A microscale thermal–electrical field-flow fractionation (ThElFFF) channel is reported for the first time and preliminary characterization results show high retention at certain operating conditions including relatively high flow rates when compared to standard microscale electrical or thermal field-flow fractionation instruments. A new design is presented that simplifies manufacturing and assembly of the prototype and that can provide both an electrical field and a high temperature gradient (∼106°C/m). Monodisperse particle retention is carried out with polystyrene nanoparticle samples to characterize the device. Retention ratios as low as 0.045 are observed with the ThElFFF instrument. Size selectivity of 1.77 was achieved for ThElFFF. The comparison with theory shows a marked deviation from the existing theory. Separation of a mixture of polystyrene particles is demonstrated for the first time using a ThElFFF system by separating 130nm carboxylated polystyrene and 209nm polystyrene particles.