Flow FFF Research Articles

Increasing the Resolution of Field-Flow Fractionation with Increasing Crossflow Gradients.

The resolution of flow field-flow fractionation (flow FFF) depends primarily on the crossflow rate and its change over time. In this work, we demonstrate a method for modulation of the crossflow rate during separation that increases the peak-to-peak resolution of the resulting fractograms. In classical FFF methods, the crossflow rate is either maintained constant or decreased during the separation of the different species. In this work, higher resolution between peaks was achieved by a novel gradient method in which the crossflow is increased briefly during separation to allow stronger retention of the later eluting peaks. We first outline the theoretical basis by which improved separation is achieved. We confirm our hypothesis by quantifying the impact of increasing crossflow on the resolution between a monoclonal antibody monomer and its high-molecular-weight aggregate. We then demonstrate that this method is applicable to two different FFF methods (AF4 and HF5) and various pharmaceutically relevant samples (monoclonal antibodies and adeno-associated viruses). Finally, we hypothesize that increasing the force perpendicular to the laminar flow as described here is broadly applicable to all FFF methods and improves the quality of FFF-based separations.

Optimization for size separation of graphene oxide sheets by flow/hyperlayer field-flow fractionation

Graphene oxide (GO)-a chemical derivative of graphene with numerous oxygen functional groups on its surface-has attracted considerable interest because of its intriguing properties in relation to those of pristine graphene. In addition to the inherent wide lateral size distribution of GO sheets arising from the typical oxidative exfoliation of graphite, control of the lateral size of GO is critical for desired GO-based applications. Herein, flow/hyperlayer field-flow fractionation (flow/hyperlayer FFF) is optimized to separate GO sheets by lateral dimensions. Optimized fractionation is achieved by investigating the influences of carrier solvent, channel thickness, and flow rate conditions on the steric/hyperlayer separation of GO sheets by flow FFF. Due to the strong hydrodynamic lift forces of extremely thin GO sheets, a thick flow FFF channel (w=350 μm) and a very low field strength are required to retain the GO sheets within the channel. GO sheets with narrow size fractions are successfully collected from two different graphite sources during flow/hyperlayer FFF runs and are examined to verify the size evolution. Considering the average lateral diameter of the GO fraction calculated on the basis of the assumption of a circular disk shape, the retention of the GO sheets is 2.2-5.0 times faster than that of spherical particles of the same diameter. This study demonstrates that through flow/hyperlayer FFF, the size distribution of GO sheets can be determined and narrow size fractions can be collected (which is desirable for GO-based applications), which are commonly influenced by the GO lateral dimension.

Supramolecular Answers to the Organic Matter Controversy

The foundation of separation science is the division or deconvolution of a mixture into constituent parts. To do so greatly informs our understanding of complex systems. Equally important is consideration of the behavior of a mixture as a whole—to consider if the whole is the sum of the parts or if individual components interact synergistically or antagonistically when combined. Application of separation science has seldom assumed greater importance than in consideration of the controversy surrounding the traditional and emergent views of the complex chemical system of natural organic matter (NOM). Analyses of simulated and actual environmental water samples using noninvasive separation techniques including flow field‐flow fractionation (flow FFF), excitation–emission matrix (EEM) fluorescence spectroscopy with parallel factor (PARAFAC) analyses, and dynamic light scattering (DLS) are discussed. The data are used to explore whether a distinct chemical category of humic (diagenetic) substances exists in NOM; whether separation of organic matter into fulvic and humic extracts has relevant meaning translatable to actual environmental water samples; whether extraction procedures alter the chemistry of NOM extracts; and, if humic and fulvic acids exist, what physicochemical property or properties make humic substances unique among other forms of NOM. A fluorescence‐based nonoperational definition of humic substances is introduced. The underexplored role of supramolecular, self‐assembled aggregation is presented as a NOM conceptual model. Responses are provided to questions raised in the great NOM debate within a supramolecular context.Core Ideas A new supramolecular conceptual model invokes strong near‐covalent hydrogen bonding. A nonoperational definition of diagenetic (humic) substances is proposed. Alkali extracts are valid, though imperfect, organic matter proxies. A mechanism for intrinsic chemical recalcitrance of natural organic matter exists. Protection is a “function” of the supramolecular “form.”

Flow optimisations with increased channel thickness in asymmetrical flow field-flow fractionation

Retention in flow field-flow fractionation (flow FFF) is generally governed by the combination of crossflow and migration flowrates. Especially for an asymmetrical flow FFF (AF4) channel in which the channel-inlet flow is divided into crossflow and outflow, the separation of low-molecular-weight proteins or macromolecules requires a relatively high crossflow rate along with a very low outflow rate for a reasonable level of resolution, which often leads to a limitation in channel pressure. In this study, the performances of AF4 with increased channel thicknesses have been investigated by adjusting the effective channel flowrates in the asymmetrical channels according to the variation of channel thickness. Four AF4 channels of different channel thicknesses (350, 490, 600, and 740 μm) were employed to examine the potential usefulness of employing a thick channel in the high-resolution separation of low-molecular-weight proteins (< 100 kDa) and to determine the relationship between higher channel thickness and the recovery of elution. Experiments showed that the ratio of crossflow rate to the effective channel flowrate should be considered in the selection of a run condition at an increased channel thickness. The study also demonstrated that a thick AF4 channel can be useful for the high-resolution separation of low-molecular-weight species such as protein aggregates without using extremely high crossflow rates.

Conductivity-Dependent Flow Field-Flow Fractionation of Fulvic and Humic Acid Aggregates

Fulvic (FAs) and humic acids (HAs) are chemically fascinating. In water, they have a strong propensity to aggregate, but this research reveals that tendency is regulated by ionic strength. In the environment, conductivity extremes occur naturally—freshwater to seawater—warranting consideration at low and high values. The flow field flow fractionation (flow FFF) of FAs and HAs is observed to be concentration dependent in low ionic strength solutions whereas the corresponding flow FFF fractograms in high ionic strength solutions are concentration independent. Dynamic light scattering (DLS) also reveals insight into the conductivity-dependent behavior of humic substances (HSs). Four particle size ranges for FAs and humic acid aggregates are examined: (1) &lt;10 nm; (2) 10 nm–6 µm; (3) 6–100 µm; and (4) &gt;100 µm. Representative components of the different size ranges are observed to dynamically coexist in solution. The character of the various aggregates observed—such as random-extended-coiled macromolecules, hydrogels, supramolecular, and micellar—as influenced by electrolytic conductivity, is discussed. The disaggregation/aggregation of HSs is proposed to be a dynamic equilibrium process for which the rate of aggregate formation is controlled by the electrolytic conductivity of the solution.

Comments on the separation efficiency of asymmetrical flow field-flow fractionation in channels of constant channel and crossflow velocities leading to constant separation efficiency

It is shown theoretically that a claim in the literature about the overall separation efficiency of asymmetrical flow FFF channels being improved by geometries that permit a uniform channel flow velocity throughout the channel length is untrue.

On the no-field method for void time determination in flow field-flow fractionation

Elution time measurements of colloidal particles injected in a symmetrical flow field-flow fractionation (flow FFF) system when the inlet and outlet cross-flow connections are closed have been performed. This no-field method has been proposed earlier for void time (and void volume) determination in flow FFF Giddings et al. (1977) [8]. The elution times observed were much larger than expected on the basis of the channel geometrical volume and the flow rate. In order to explain these discrepancies, a flow model allowing the carrier liquid to flow through the porous walls toward the reservoirs located behind the porous elements and along these reservoirs was developed. The ratio between the observed elution time and expected one is found to depend only on a parameter which is a function of the effective permeability and thickness of the porous elements and of the channel thickness and length. The permeabilities of the frits used in the system were measured. Their values lead to predicted elution times in reasonable agreement with experimental ones, taking into account likely membrane protrusion inside the channel on system assembly. They comfort the basic feature of the flow model, in the no-field case. The carrier liquid mostly bypasses the channel to flow along the system mainly in the reservoir. It flows through the porous walls toward the reservoirs near channel inlet and again through the porous walls from the reservoirs to the channel near channel outlet before exiting the system. In order to estimate the extent of this bypassing process, it is desirable that the hydrodynamic characteristics of the permeable elements (permeability and thickness) are provided by flow FFF manufacturers. The model applies to symmetrical as well as asymmetrical flow FFF systems.

Development of a Multilane Channel System for Nongel-Based Two-Dimensional Protein Separations Using Isoelectric Focusing and Asymmetrical Flow Field-Flow Fractionation

A dual purpose multilane channel system to carry out isoelectric focusing (IEF) and asymmetrical flow field-flow fractionation (IEF-AFlFFF or IEF-AF4) was developed for the high-speed fractionation of a proteome in two dimensions (2D): isoelectric point (pI) and hydrodynamic diameter (d(s)). Separation of proteins is initially achieved by differences in pI using IEF in an open thin segment, which is formed by interconnecting the beginning part of six parallel flow FFF channels in the lateral direction. After IEF, each protein pool of a different pI interval is simultaneously separated in an orthogonal direction by d(s) in six individual AF4 channels. The developed IEF-AF4 multilane channel system provides ultimate nongel, elution based, and 2D protein separation at an improved separation speed; the entire separation can be processed within 30 min, compared to approximately 3 h with the previously developed capillary isoelectric focusing-hollow fiber FlFFF (CIEF-HFFlFFF or CIEF-HF5) (Kang, D.; Moon, M. H. Anal. Chem. 2006, 78, 5789-5798) or approximately 36 h with 2D-polyacryamide gel electrophoresis (2D-PAGE). An initial evaluation of IEF-AF4 was performed to investigate the influence of ampholyte concentration and IEF voltage on the separation of standard protein mixtures.

Easy Monitoring, by Flow FFF, of the Behavior of Nanoparticles Formed from Copolymers of Sulfopropylmethacrylate and Methylmethacrylate

The size and size distribution of nanoparticles are generally important for the characterization of these materials, but are particularly needed to determine their suitability for specific applications. When nanoparticles are obtained by micelle formation from synthetic copolymers their size and structure depend on the copolymer composition and charge. Minor changes in the macromolecule primary structure may lead to dramatic changes in the aggregation behavior, hence in the nanoparticle size and size distribution. While particle diameter may be accurately measured by various sizing methods, determination of the size distribution requires a methodology which combines an adequate resolving power with size measurement capabilities. Flow FFF has shown to be able to perform both tasks in a single run. Using a very small amount of sample it yields accurate size distribution curves of most various colloidal systems. For this reason it was used here for the analysis of two copolymers of sulfopropyl methacrylate and methylmethacrylate differing for the copolymerization process. Size analysis of these samples showed a dramatic difference in their capability of forming micellar aggregates as well as in the characteristic response to the solution ionic strength.

Variation in colloidal chromophoric dissolved organic matter in the Damariscotta Estuary, Maine

The relationship between chromophoric dissolved organic matter (CDOM), colloidal DOM (.1 kDa), and phytoplankton biomass in marine waters of the Damariscotta estuary, Maine, was studied using flow field‐flow fractionation (flow FFF). The goal was to determine whether temporal variability in CDOM in this region is due in part to rapid cycling of marine colloids. CDOM absorption of filtered (0.2 µm) seawater correlated positively with colloidal absorbance (r2 = 0.772, p = 0.021). We observed changes in colloidal size spectra and CDOM optical properties associated with phytoplankton biomass. In particular, a significant positive correlation was found between the &lt; 18 kDa colloidal fraction and chlorophyll a (Chl a) (r2 = 0.819, p = 0.013). While we did not find significant relationships between Chl a and either CDOM absorption or the spectral slope of filtered seawater (r2 and p values of 0.079, 0.590 and 0.111, 0.519, respectively), we did observe patterns of higher CDOM absorption and spectral slopes following periods of increased phytoplankton biomass. The spectral slope of colloidal material often was significantly lower than that of filtered seawater and normally varied significantly with colloidal size. These findings show that colloidal processes can contribute to CDOM optical signatures in coastal waters.

Application of a high-performance liquid chromatography fluorescence detector as a nephelometric turbidity detector following Field-Flow Fractionation to analyse size distributions of environmental colloids

A new operation mode for HPLC-type fluorescence detectors is presented and evaluated using synthetic and environmental particles in the colloidal size range. By applying identical wavelengths for excitation and emission a nephelometric turbidity or single angle light scattering detector is created which can be easily coupled to flow or sedimentation Field-Flow Fractionation (Flow FFF or Sed FFF) for the analysis of colloidal dispersions. The results are compared with standard UV–vis detection methods. Signals obtained are given as a function of particle size and selected detection wavelength. Conclusions can be drawn which affect the current practice of FFF but also for other techniques as groundwater sampling and laboratory column experiments when turbidity is measured in nephelometric mode and in small sample volumes or at low flow rates.

Investigation of nano-colloid transport in UF membranes using flow field-flow fractionation (flow FFF) and an irreversible thermodynamic transport model

Membrane fouling has been associated with the size of solutes, i.e., macromolecules and colloids in membrane filtration. Colloidal fouling propensity was investigated in terms of transport characteristics by the thermodynamic transport model combined with concentration polarization and flow field-flow fractionation (flow FFF). Transport characteristics in membranes were focused on, especially nano-colloid fouling in UF membranes. Whey protein and 30 nm of latex microsphere suspensions were used as test solutions. Transport parameters were determined in terms of mass transfer coefficient ( k), solute permeability ( P m ), and reflection coefficient (σ) by non-linear estimation. The flow FFF provided a consistent explanation for the nano-colloid transport, depending on the physicochemical interactions between membrane and solute through the transport parameters determined from filtration tests. The second and third moments, variance and skewness were expected to be very useful quantitative information on the membrane fouling propensity of a certain solute.

Characterization of Colloidal and Humic-Bound Ni and U in the “Dissolved” Fraction of Contaminated Sediment Extracts

The dissolved phase of environmental aqueous samples is generally defined by filtration at 0.2 microm or even 0.45 microm. However, it is also acknowledged that colloids <0.2 microm suspended in the aqueous phase can be important for determining contaminant availability and mobility. We have used flow field-flow fractionation (FI FFF) and size exclusion chromatography (SEC) coupled to UV-absorbance (UVA) and inductively coupled plasma mass spectrometry (ICP-MS) to study the dissolved organic matter (DOM) and colloidal binding of U and Ni in water extracts of sediments collected from a contaminated area of the Savannah River Site, a U.S. Department of Energy former nuclear materials production and processing facility, near Aiken, SC. High-performance SEC-UVA-ICP-MS was well-suited to the separation of DOM overthe molecular weight (MW) range of approximately 200-7000 Da. The ICP-MS element specific data indicated that a significant fraction of U was associated with DOM. Uranium exhibited a bimodal distribution and the other fraction was greater than the exclusion limit for the column and coeluted with Al. Flow FFF was used to size this fraction as colloidal with an approximate effective spherical diameter of 0.09-0.12 microm. Element specific ICP-MS data confirmed that U and Al were associated with the colloidal phase. High-field FI FFF was also applicable to sizing DOM but resolution was poorer than SEC. The results of this study suggest that "dissolved" U at this site is predominantly either complexed by DOM or bound to a colloidal fraction while Ni is predominately present as labile complexes or the free cation and, therefore, potentially bioavailable.

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.

Analysis of whole bacterial cells by flow field-flow fractionation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The purpose of this study is to develop a novel bacterial analysis method by coupling the flow field-flow fractionation (flow FFF) separation technique with detection by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The composition of carrier liquid used for flow FFF was selected based on retention of bacterial cells and compatibility with the MALDI process. The coupling of flow FFF and MALDI-TOF MS was demonstrated for P. putida and E. coli. Fractions of the whole cells were collected after separation by FFF and further analyzed by MALDI-MS. Each fraction, collected over different time intervals, corresponded to different sizes and possibly different growth stages of bacteria. The bacterial analysis by flow FFF/MALDI-TOF MS was completed within 1 h with only preliminary optimization of the process.

HPLC of humic substances fractionated by Flow FFF

AbstractThis communication reports a study of the effect of ionic strength and electrolyte composition on fractions, separately collected by flow FFF, of a mixture of humic substances. Reverse phase HPLC analysis of three early eluting fractions suggests that the components released by the column behave as organic acids. The baseline‐resolved peaks of the first two fractions, subject to higher retention in solutions of lower pH and/or higher polarity, substantiate this suggestion. The fraction with larger components, as measured by flow FFF, also appears to contain acidic species. Their retention level, however, may not be accurately modulated by varying the mobile phase properties as these species are either totally retained in acidic phases or released before the void peak at pH ≥ 4.2. Besides showing the effective separation achieved in the flow FFF channel, this study reveals the pronounced difference in the physicochemical properties of some components of a humic mixture even with very close particle size.

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.

Flow field-flow fractionation as an analytical technique to rapidly quantitate membrane fouling

The initial fouling behavior of a clean membrane surface was studied using flow field-flow fractionation (flow FFF), an analytical technique typically used to separate and characterize macromolecules and particulates. This work represents the first time flow FFF has been used to quantitatively evaluate membrane performance. Flow FFF is an ideal tool for expeditiously studying sample–membrane interactions for the following reasons: membranes can be quickly installed into the flow FFF channel, each analysis requires only microgram amounts of sample, and sample–membrane interactions can be rapidly quantitated for different flowrates and solution compositions. Suwannee River humic acids were used as a probe to investigate the initial fouling of an XLE reverse osmosis membrane and an NF-200 nanofiltration membrane. Flow FFF was successfully used to quantitate the fouling of each membrane and to demonstrate that the majority of sample loss was due to irreversible adsorption. The fouling on both membranes was enhanced by increasing the flowrate perpendicular to the membrane surface and by adding calcium ions to the solution. The NF-200 membrane was more resistant than the XLE membrane to fouling in the presence of calcium ions, whereas, the fouling resistance of both membranes improved to similar levels with the addition of EDTA to a solution containing calcium ions.

Separation and Characterization of Barley Starch Polymers by a Flow Field‐Flow Fractionation Technique in Combination with Multiangle Light Scattering and Differential Refractive Index Detection

ABSTRACTFlow field‐flow fractionation (flow FFF) with frit inlet and frit outlet mode (FIFO) was coupled online to multiangle light scattering (MALS) and refractive index (RI) detectors to investigate the molecular characteristics of normal and zero amylose barley starch polymers. Application of two different cross‐flows, 0.35 mL/min followed by 0.1 mL/min, and constant channel and frit flows of 0.1 and 1.0 mL/min, respectively, permitted a complete separation of amylose and amylopectin. The improved signals from the detectors due to application of the FIFO mode enabled the proper characterization of the small molecular weight species, as well as significantly enhanced the reproducibility of the measurements. The weight‐average molecular weight (Mw) and zaverage root‐mean‐square (RMS) radii of gyration (Rg) values for amylose and amylopectin in the normal starch samples were 2.3 × 106 and 280 × 106, and 107 and 260 nm, respectively. The Mw and Rg of amylopectin in the zero amylose starch samples were 360 × 106 and 267 nm, respectively. The slopes (α) obtained by plotting log Mw versus log Rg for amylose and amylopectin were 0.6 and 0.3, respectively. These results are in good agreement with the theoretical prediction of the molecular conformation of amylose and amylopectin.

Effects of ionic strength and electrolyte composition on the aggregation of fractionated humic substances studied by flow field-flow fractionation

A sample of humic substances (HS) from Aldrich Co. has been studied by flow field-flow fractionation. The effect of the injected mass on retention, comparable to that previously found for synthetic water-soluble polyelectrolytes, suggests that the components present in this sample as Na salts are highly charged in water. Charges are, however, screened when a simple electrolyte is added to the carrier liquid. The electrolyte also appears to induce molecular aggregation to an extent that depends on the type of the metal ion and on its concentration. Investigation of the effect of the solution ionic strength and composition on fractions separately collected from the flow FFF channel confirms the aggregating effect of the salt added to the solubilizing medium and reveals a stronger tendency to particle aggregation by the HS in the presence of divalent cations. This finding seems common to all the fractions except that containing particles of lower size that appear to respond only to the ionic strength. A comparative analysis of the ionic strength effect on some fractions and on the unfractionated sample seems to support the hypothesis that, in the latter, the aggregation process occurs through a non-size-specific pathway. Therefore, in a broadly-dispersed humic mixture in solution of simple electrolytes, the size distribution is likely to be determined by random interactions of species of different size.