Field-flow Fractionation Techniques Research Articles

Delicate Analysis of Interacting Proteins and Their Assemblies by Flow Field-Flow Fractionation Techniques.

We study the efficiency of several asymmetrical flow field-flow fractionation (AF4) techniques to investigate self-associating wheat gluten proteins. We compare the use of a denaturing buffer including sodium dodecyl sulfate (SDS) and a mild chaotropic solvent, water/ethanol, as the eluent, on a model gluten sample. Through a thorough analysis of the data obtained from coupled light scattering detectors and with the identification of molecular composition of the eluted protein, we evidence coelution events in several conditions. We show that the focus step used in conventional AF4 with the SDS buffer leads to the formation of aggregates that coelute with monomeric proteins. By contrast, a frit-inlet device enables the fractionation of individual wheat proteins in the SDS buffer. Interestingly conventional AF4, using water/ethanol as eluent, is an effective method for fractionating gluten proteins and their complex dynamic assemblies, which involve weak forces and are composed of both monomeric and polymeric proteins.

Isolation of Carbon Black from Soils by Dispersion for Analysis: Quantitation and Characterization by Field Flow Fractionation Techniques.

In the present work, a procedure based on a dispersive medium for carbon black (CB) isolation from soil samples for analysis was proposed for the first time. Polymeric and biological dispersants and a sequential use of both dispersants were assayed. Asymmetrical flow field flow fractionation with dynamic light scattering detector (AF4-DLS) and sedimentation field flow fractionation with multi-angle light scattering detector (SdF3-MALS) were used for CB quantitation and characterization in the achieved dispersions. Soil samples contaminated with CB were processed, and CB isolation depended on the solid size distribution and composition and dispersant nature. More quantitative isolations were achieved for the four soils treated by the biological dispersant. As the organic matter percentage is higher in soil, the CB isolation was better, varying between 75 and 99% with standard deviation (s) ⩽ 2% for all soils. A soil contaminated with a CB-based pigment paste was analyzed, achieving (99 ± 2)% expressed as expanded uncertainty (K = 2) of dispersive isolation by the biological dispersant, and the sampling was scaled to 250 g of soil with positive results. The procedure was completed by CB recovery to obtain a solid residue able to be reused if necessary. For the filter-aided recovery step, different membranes (fiberglass, nylon, and Teflon) with a pore size between 0.1 and 5 μm were tested. The quantitation of the CB retained in the filter was measured by diffuse reflectance spectroscopy. Teflon (0.10 μm) provided better results for CB recovery, and its re-dispersion was also studied with suitable results. Determination of CB from the filters by diffuse reflectance spectrometry provided the same results than AF4 for CB dispersions.

Field-Flow Fractionation in Molecular Biology and Biotechnology.

Field-flow fractionation (FFF) is a family of single-phase separative techniques exploited to gently separate and characterize nano- and microsystems in suspension. These techniques cover an extremely wide dynamic range and are able to separate analytes in an interval between a few nm to 100 µm size-wise (over 15 orders of magnitude mass-wise). They are flexible in terms of mobile phase and can separate the analytes in native conditions, preserving their original structures/properties as much as possible. Molecular biology is the branch of biology that studies the molecular basis of biological activity, while biotechnology deals with the technological applications of biology. The areas where biotechnologies are required include industrial, agri-food, environmental, and pharmaceutical. Many species of biological interest belong to the operational range of FFF techniques, and their application to the analysis of such samples has steadily grown in the last 30 years. This work aims to summarize the main features, milestones, and results provided by the application of FFF in the field of molecular biology and biotechnology, with a focus on the years from 2000 to 2022. After a theoretical background overview of FFF and its methodologies, the results are reported based on the nature of the samples analyzed.

Electrochemical treatment for leachate membrane retentate: Performance comparison of electrochemical oxidation and electro-coagulation technology.

With the widespread use of membrane in advanced treatment of leachate, China produces a large amount of leachate membrane retentate (LMR) (≈23.4 million tons) annually, which is usually treated by incineration or recirculation in engineering, but these technologies have many drawbacks. LMR is suitable for electrochemical treatment due to its high electrical conductivity. This study compared the performances of electrochemical oxidation (EO) and electro-coagulation (EC) technology on LMR treatment under different experimental conditions, including anode material, current density, initial pH and reaction time. We found that EO optimal conditions achieved 70.1%, 83.1%, 78.7%, 98.7%, and 69.7% removal of total organic carbon (TOC), UV absorption (at 254nm), chromaticity, ammonia nitrogen (NH3-N), and total nitrogen (TN), respectively. Compared with EO, EC exhibited a similar removal ability for orgainics and better removals of chroma, but much less performance for removing nitrogen pollutants in the same reaction time, that is, removals of NH3-N and TN were only 31.5% and 36.2%, respectively. Meanwhile, EC showed much higher instantaneous current efficiency of COD than EO under its optimal reaction time (120min). In addition, the UV-Vis spectra and 3D fluorescence spectra indicated that EO exhibited relatively outstanding performance in decomposing dissolved organic matter (DOM) with rather complicated structures than EC. Also, the flow field-flow fractionation technique demonstrated that EO preferentially destroy humic-like, large molecular weight DOM, and converting them to smaller molecules, which resulted in more volatile organic compounds in EO samples than EC samples. While EC had little selectivity in the removal of organics, except humic-like DOM with relative small molecular. These findings can provide a theoretical basis for the electrochemical treatment of LMR.

A Label-Free Cell Sorting Approach to Highlight the Impact of Intratumoral Cellular Heterogeneity and Cancer Stem Cells on Response to Therapies.

Cancer stem cells play a crucial role in tumor initiation, metastasis, and resistance to treatment. Cellular heterogeneity and plasticity complicate the isolation of cancer stem cells. The impact of intra-tumor cellular heterogeneity using a label-free approach remains understudied in the context of treatment resistance. Here, we use the sedimentation field–flow fractionation technique to separate, without labeling, cell subpopulations of colorectal cancer cell lines and primary cultures according to their biophysical properties. One of the three sorted cell subpopulations exhibits characteristics of cancer stem cells, including high tumorigenicity in vivo and a higher frequency of tumor-initiating cells compared to the other subpopulations. Due to its chemoresistance, two- and three-dimensional in vitro chemosensitivity assays highlight the therapeutic relevance of this cancer stem cell subpopulation. Thus, our results reveal the major implication of intra-tumor cellular heterogeneity, including cancer stem cells in treatment resistance, thanks to our label-free cell sorting approach. This approach enables—by breaking down the tumor—the study the individualized response of each sorted tumor cell subpopulation and to identify chemoresistance, thus offering new perspectives for personalized therapy.

Methodology of measurement of ionic strength based on field-flow fractionation

In this report, we put forward an experimental method to determine the ionic strength of an aqueous solution. To this end, we have developed a theory of ionic strength I expressed in terms of the retention ratios in field-flow fractionation (FFF) as I1/2=κ(1−R)/(1−Ro)−ε−1/2. Here R is a measured retention ratio using an FFF technique, for instance, sedimentation FFF (SdFFF), and Ro is the sterically-corrected standard retention ratio as given by the standard retention theory (SRT) for a latex particle system of diameter d. For a standard latex system with known d (or Ro) and I, we can construct a linear calibration of I1/2 against (1−R)/(1−Ro). Therefore, if we measure the retention ratio R of a carrier liquid of which ionic strength is of interest, then we will be able to estimate the ionic strength from the calibration curve thus built. In this paper, we have demonstrated the relation of I1/2with respect to (1−R)/(1−Ro) for the polystyrene latex systems of which information on R, Ro, and I is available from Ref. [1].

Physical characterization of biomedical magnetic nanoparticles using multi-detector centrifugal field-flow fractionation

Abstract The unique magnetic properties of magnetic nanoparticles (MNP) combined with their small size already led to numerous medical applications. Accurate determination of their magnetic properties is a key requirement enquired by users, that is impeded by the ever-present distribution of MNP sizes. Field flow fractionation (FFF) techniques may help to overcome these limitations by first separating the particles before characterization. In this study, we demonstrate the use of centrifugal FFF coupled to online detectors for fractionation, structural, and magnetic characterization of MNP. The primary goal is to establish a reproducible centrifugal FFF (CF3) method for MNP fractionation We show that CF3 has the same capability as other FFF techniques in resolving the bimodal hydrodynamic size distribution present in the commercial MNP system Resovist® but is faster and more straightforward through its technical approach.

New Advances and Applications in Field-Flow Fractionation.

Field-flow fractionation (FFF) is a family of techniques that was created especially for separating and characterizing macromolecules, nanoparticles, and micrometer-sized analytes. It is coming of age as new nanomaterials, polymers, composites, and biohybrids with remarkable properties are introduced and new analytical challenges arise due to synthesis heterogeneities and the motivation to correlate analyte properties with observed performance. Appreciation of the complexity of biological, pharmaceutical, and food systems and the need to monitor multiple components across many size scales have also contributed to FFF's growth. This review highlights recent advances in FFF capabilities, instrumentation, and applications that feature the unique characteristics of different FFF techniques in determining a variety of information, such as averages and distributions in size, composition, shape, architecture, and microstructure and in investigating transformations and function.

Adeno-associated Virus Virus-like Particle Characterization via Orthogonal Methods: Nanoelectrospray Differential Mobility Analysis, Asymmetric Flow Field-Flow Fractionation, and Atomic Force Microscopy.

Adeno-associated virus (AAV)-based virus-like particles (VLPs) are thriving vectors of choice in the biopharmaceutical field of gene therapy. Here, a method to investigate purified AAV serotype 8 (AAV8) batches via a nanoelectrospray gas-phase mobility molecular analyzer (nES GEMMA), also known as an nES differential mobility analyzer, is presented. Indeed, due to AAV’s double-digit nanometer scale, nES GEMMA is an excellently suited technique to determine the surface-dry particle size termed electrophoretic mobility diameter of such VLPs in their native state at atmospheric pressure and with particle-number-based detection. Moreover, asymmetric flow field-flow fractionation (AF4, also known as AFFFF) and atomic force microscopy (AFM) techniques were employed as orthogonal techniques for VLP characterization. In addition, AF4 was implemented to size-separate as well as to enrich and collect fractions of AAV8 VLPs after inducing analyte aggregation in the liquid phase. Bionanoparticle aggregation was achieved by a combination of heat and shear stress. These fractions were later analyzed with nES GEMMA (in the gas phase) and AFM (on a solid surface). Both techniques confirm the presence of dimers, trimers, and putative VLP oligomers. Last, AFM reveals even larger AAV8 VLP aggregates, which were not detectable by nES GEMMA because their heterogeneity combined with low abundance was below the limit of detection of the instrument. Hence, the combination of the employed orthogonal sizing methods with the separation technique AF4 allow a comprehensive characterization of AAV8 VLPs applied as vectors.

Organic Carbon Linkage with Soil Colloidal Phosphorus at Regional and Field Scales: Insights from Size Fractionation of Fine Particles.

Nano and colloidal particles (1-1000 nm) play important roles in phosphorus (P) migration and loss from agricultural soils; however, little is known about their relative distribution in arable crop soils under varying agricultural geolandscapes at the regional scale. Surface soils (0-20 cm depth) were collected from 15 agricultural fields, including two sites with different carbon input strategies, in Zhejiang Province, China, and water-dispersible nanocolloids (0.6-25 nm), fine colloids (25-160 nm), and medium colloids (160-500 nm) were separated and analyzed using the asymmetrical flow field flow fractionation technique. Three levels of fine-colloidal P content (3583-6142, 859-2612, and 514-653 μg kg-1) were identified at the regional scale. The nanocolloidal fraction correlated with organic carbon (Corg) and calcium (Ca), and the fine colloidal fraction with Corg, silicon (Si), aluminum (Al), and iron (Fe). Significant linear relationships existed between colloidal P and Corg, Si, Al, Fe, and Ca and for nanocolloidal P with Ca. The organic carbon controlled colloidal P saturation, which in turn affected the P carrier ability of colloids. Field-scale organic carbon inputs did not change the overall morphological trends in size fractions of water-dispersible colloids. However, they significantly affected the peak concentration in each of the nano-, fine-, and medium-colloidal P fractions. Application of chemical fertilizer with carbon-based solid manure and/or modified biochar reduced the soil nano-, fine-, and medium-colloidal P content by 30-40%; however,the application of chemical fertilizer with biogas slurry boosted colloidal P formation. This study provides a deep and novel understanding of the forms and composition of colloidal P in agricultural soils and highlights their spatial regulation by soil characteristics and carbon inputs.

Characterisation of selenium and tellurium nanoparticles produced by Aureobasidium pullulans using a multi-method approach

Aureobasidium pullulans was grown in liquid culture media amended with selenite and tellurite and selenium (Se) and tellurium (Te) nanoparticles (NPs) were recovered after 30 d incubation. A separation method was applied to recover and characterise Se and Te NPs by asymmetric flow field flow fractionation (AF4) with online coupling to multi-angle light scattering (MALS), ultraviolet visible spectroscopy (UV-Vis), and inductively coupled plasma mass spectrometry (ICP-MS) detectors. Additional characterisation data was obtained from transmission electron microscopy (TEM), and dynamic light scattering (DLS). Solutions of 0.2% Novachem surfactant and 10 mM phosphate buffer were compared as mobile phases to investigate optimal AF4 separation and particle recovery using Se-NP as a model sample. 88% recovery was reported for 0.2% Novachem solution, compared with 50% recovery for phosphate buffer. Different crossflow (Cflow) rates were compared to further investigate optimum separation, with recoveries of 88% and 30% for Se-NPs, and 90% and 29% for Te-NPs for 3.5 mL min−1 and 2.5 mL min−1 respectively. Zeta-potential (ZP) data suggested higher stability for NP elution in Novachem solution, with increased stability attributed to minimised NP-membrane interaction due to PEGylation. Detection with MALS showed monodisperse Se-NPs (45–90 nm) and polydisperse Te-NPs (5–65 nm).Single particle ICP-MS showed mean particle diameters of 49.7 ± 2.7 nm, and 135 ± 4.3 nm, and limit of size detection (LOSD) of 20 nm and 45 nm for Se-NPs and Te-NPs respectively. TEM images of Se-NPs and Te-NPs displayed a spherical morphology, with the Te-NPs showing a clustered arrangement, which suggested electrostatic attraction amongst neighbouring particles. Particle hydrodynamic diameters (dH) measured with dynamic light scattering (DLS) further suggested monodisperse Se-NPs and polydisperse Te-NPs distributions, showing good agreement with AF4-MALS for Se-NPs, but suggests that the Rg obtained from AF4-MALS for Te-NP was unreliable. The results demonstrate a complementary application of asymmetric flow field-flow fractionation (AF4), ICP-MS, light scattering, UV-Vis detection, and microscopic techniques to characterise biogenic Se and Te NPs.

Limitations of Nanoparticles Size Characterization by Asymmetric Flow Field\u2011Fractionation Coupled with Online Dynamic Light Scattering

The development of reliable protocols suitable for the characterisation of the physical properties of nanoparticles in suspension is becoming crucial to assess the potential biological as well as toxicological impact of nanoparticles. Amongst sizing techniques, asymmetric flow field flow fractionation (AF4) coupled to online size detectors represents one of the most robust and flexible options to quantify the particle size distribution in suspension. However, size measurement uncertainties have been reported for on-line dynamic light scattering (DLS) detectors when coupled to AF4 systems. In this work we investigated the influence of the initial concentration of nanoparticles in suspension on the sizing capability of the asymmetric flow field-flow fractionation technique coupled with an on-line dynamic light scattering detector and a UV–Visible spectrophotometer (UV) detector. Experiments were performed with suspensions of gold nanoparticles with a nominal diameter of 40 nm and 60 nm at a range of particle concentrations. The results obtained demonstrate that at low concentration of nanoparticles, the AF4-DLS combined technique fails to evaluate the real size of nanoparticles in suspension, detecting an apparent and progressive size increase as a function of the elution time and of the concentration of nanoparticles in suspension.

Fast and effective chromatographic separation of polymersomes from proteins by multimodal chromatography

Artificial vesicles made of block copolymers, so-called polymersomes, represent a versatile chassis for the creation of functionalized nanocompartments with a wide range of biotechnological applications. The specific application depends on the biomolecules – usually proteins – that are positioned in the interior, in the membrane or on the surface of the vesicles. However, not all added proteins are integrated into the vesicles during the usual manufacturing processes of polymersomes. Excess proteins must therefore be removed. The separation techniques currently used for this, however, are associated with decisive disadvantages, such as damaged vesicles, long process times, or small sample volumes that can be processed. To overcome these drawbacks, we investigated the applicability of Capto™ Core 700 resin for polymersome purification. Polymersomes were not damaged or otherwise affected by passage through the column verified by hollow fiber flow field flow fractionation technique. Using three proteins with divergent physico-chemical properties as examples, it was demonstrated that different types of unentrapped proteins were efficiently removed from polymersome dispersions. The dynamic binding capacities in the presence of polymersomes varied between 9.5 and 16.5 mg per mL resin for the proteins applied. The technique can be used for small and large sample volumes alike. In addition, it can be used without special laboratory equipment. This adds a new and easy-to-use purification method for polymer vesicles to the repertoire that will also facilitate the large-scale production of functionalized polymersomes.

Characterisation of the Interaction among Oil-In-Water Nanocapsules and Mucin.

Mucins are glycoproteins present in all mucosal surfaces and in secretions such as saliva. Mucins are involved in the mucoadhesion of nanodevices carrying bioactive molecules to their target sites in vivo. Oil-in-water nanocapsules (NCs) have been synthesised for carrying N,N′-(di-m-methylphenyl)urea (DMTU), a quorum-sensing inhibitor, to the oral cavity. DMTU-loaded NCs constitute an alternative for the treatment of plaque (bacterial biofilm). In this work, the stability of the NCs after their interaction with mucin is analysed. Mucin type III from Sigma-Aldrich has been used as the mucin model. Mucin and NCs were characterised by the multi-detection asymmetrical flow field-flow fractionation technique (AF4). Dynamic light scattering (DLS) and ζ-potential analyses were carried out to characterise the interaction between mucin and NCs. According to the results, loading DMTU changes the conformation of the NC. It was also found that the synergistic interaction between mucin and NCs was favoured within a specific range of the mucin:NC ratio within the first 24 h. Studies on the release of DMTU in vitro and the microbial activity of such NCs are ongoing in our lab.

Stability of melamine‐formaldehyde (MF) resin under different pH and ionic strength values

Melamine and melamine‐formaldehyde (MF) resin are both commonly used materials in food technology. It is shown in this work that the stability of aqueous solutions of MF resins during storage is strongly influenced by the ionic strength and the pH value of the suspending medium. Aggregation phenomena, polymerization, or disintegration of MF resin particles can take place. The critical aggregation concentration (CAC = 1.67 × 10⁻² M KNO₃) and the rate constant kₐₚₚ (1.24 × 10⁻²³ ml/s) for the bimolecular process of aggregation are determined. When the electrolyte concentration is very close to the CAC, the aggregation is slow. By increasing the electrolyte concentration the aggregation process is faster. In high values of ionic strength the aggregation happens almost immediately. At low pH values aggregation phenomena and split of the MF in smaller particles can influence the stability of MF resin particles. For this study, field flow fractionation technique was used. PRACTICAL APPLICATIONS: The present work investigates the role of ionic strength and pH on the stability of melamine. It is shown that aggregation phenomena, polymerization, or disintegration of MF resin particles can take place. Both, aggregation phenomena and split of the MF in smaller particles can influence the stability of MF resin particles at low pH values. These results show that dishes and kitchen utensils made of melamine resins could release melamine when used for acidic foods. The results of this work could be of great importance for both consumers and industry, since they concern preservation, construction, manufacturing, and use of common housewares.

Open-tubular radially cyclical electric field-flow fractionation (OTR-CyElFFF): an online concentric distribution strategy for simultaneous separation of microparticles.

An open-tubular radially cyclical electric field-flow fractionation technique which achieves the online separation of microparticles in a functional annular channel is proposed in this study. The system was set up by using a stainless steel tube and a platinum wire modified with ionic liquid/mesoporous silica materials as the external and internal electrodes. The feasibility for online separation of various particles was experimentally demonstrated. Particles in the channel were affected by a radial electric field and field-flow fractionation (FFF). On the cross section, different particles showed distinctive migration distances depending on their own properties and the different magnitudes of forces being exerted. The same kind of particles form an annular distribution within the same annulus while different particles form annular distributions at varied concentric annuli through electrophoresis. Under a laminar flow of FFF, different sizes of particles formed a conical arrangement within the annular separation channel. With the joint influence of electric field and flow field, different trajectories were obtained and the particles were eventually separated. Voltage, frequency and duty cycle value are the main parameters affecting the separation of particles. By adjusting these parameters, particles migrate in a zigzag trajectory on one side of the electrodes (mode I) and reach both sides of the electrodes (mode II). Six polystyrene particles were completely separated with high resolution within several minutes. Our system offers numerous advantages of label-free, high-resolution and online separation without tedious operations, and it is a promising tool for the effective separation of various micro-objects.

Terminology of separation methods (IUPAC Recommendations 2017)

Abstract Recommendations are given concerning the terminology of methods of separation in analytical chemistry, including chromatography, electromigration techniques, and field-flow fractionation and related techniques.

Asymmetrical flow field-flow fractionation coupled with a liquid waveguide capillary cell for monitoring natural colloids in groundwater

This paper describes an asymmetrical flow field-flow fractionation (AF4) technique combined with a flow-through cell having a long optical path length, called a liquid waveguide capillary cell (LWCC). The performance of the AF4-LWCC system for separation and spectrophotometric detection of nanoparticles was examined using size-standard silver nanoparticles. The limit of detection for silver nanoparticles of 30 and 60 nm diameter was highly improved to 2.5 and 7.8 μg L−1 (from 220 and 850 μg L−1, respectively) by applying a 100-cm LWCC, compared to a commercial UV/Vis detector. This improvement of detection sensitivity enabled investigating natural nanoparticles in granitic groundwater extracted from an underground research tunnel in the Republic of Korea. Only a small injection volume (0.5 mL) of groundwater was required to obtain the size distributions of trace amounts of natural colloids without pre-concentration. This study demonstrates that the proposed AF4-LWCC system could be applied as a convenient and efficient tool for detection and size-determination of aquatic nanoparticles at trace-level concentrations.

Pitfalls in Size Characterization of Soft Particles by Dynamic Light Scattering Online Coupled to Asymmetrical Flow Field-Flow Fractionation.

An asymmetrical flow field-flow fractionation (AF4) technique coupled to a multiangle light scattering (MALS) detector with an embedded dynamic light scattering (DLS) module was introduced to study the size characteristics and shape of soft particles of various size and type: polystyrene nanosphere size standards, lipid droplets (LDs), and large unilamellar vesicles (LUVs). A range of flow velocities through the LS detector, at which accurate hydrodynamic size can be extracted from the DLS in flow mode, was studied since the particles subjected to a longitudinal flow exhibit not only the Brownian motion due to diffusion but also the translational movement. In addition, the impact of the longitudinal flow velocity on the shape of the artificial LUV of two different sizes and two different compositions was studied by MALS. For comparison, the conventional batch DLS and static light scattering (SLS) experiments without prior sample separation by size were performed. From a combination of batch and flow light scattering results, we concluded that the passage flow velocities at the detector used in this study, 0.2, 0.5, and 1 mL/min, have no significant impact on the shape of spherical vesicles; however, the flow DLS experiments give accurate hydrodynamic radius (Rh) only at the lowest investigated passage flow rate at the detector (0.2 mL/min). With increasing rate of passage flow at the DLS detector, the error in the accuracy of the Rh determination rapidly increases. The error in Rh depends solely on the detector flow rate and particle size but not on the type of the soft particle.

Use of electrothermal atomic absorption spectrometry for size profiling of gold and silver nanoparticles

Electrothermal atomic absorption spectrometry (ETAAS) was applied to investigate the atomization behaviors of gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) in order to relate with particle size information. At various atomization temperatures from 1400 °C to 2200 °C, the time-dependent atomic absorption peak profiles of AuNPs and AgNPs with varying sizes from 5 nm to 100 nm were examined. With increasing particle size, the maximum absorbance was observed at the longer time. The time at maximum absorbance was found to linearly increase with increasing particle size, suggesting that ETAAS can be applied to provide the size information of nanoparticles. With the atomization temperature of 1600 °C, the mixtures of nanoparticles containing two particle sizes, i.e., 5 nm tannic stabilized AuNPs with 60, 80, 100 nm citrate stabilized AuNPs, were investigated and bimodal peaks were observed. The particle size dependent atomization behaviors of nanoparticles show potential application of ETAAS for providing size information of nanoparticles. The calibration plot between the time at maximum absorbance and the particle size was applied to estimate the particle size of in-house synthesized AuNPs and AgNPs and the results obtained were in good agreement with those from flow field-flow fractionation (FlFFF) and transmission electron microscopy (TEM) techniques. Furthermore, the linear relationship between the activation energy and the particle size was observed.