Geometric scaling effects on instrumental plate height in field flow fractionation

This paper examines geometric scaling models for field flow fractionation systems to understand how channel dimensions affect resolution and retention. Specifically, the changing contribution of the instrumental plate height during miniaturization of field flow fractionation (FFF) systems is reported. The work is directed towards determining the optimal geometrical parameters for miniaturization of field flow fractionation systems. The experimental relationship between channel height in FFF systems and instrumental plate heights is reported. FFF scaling models are modified to: (i) better clarify the dependence of plate height and resolution on channel height in FFF and (ii) include a more complete geometrical scaling analysis and model comparison in the low retention regime. Electrical field flow fractionation has been shown to benefit from miniaturization, so this paper focuses on that subtype, but surprisingly, the results also indicate the possibility of improvement in performance with miniaturization of other field flow fractionation systems including general FFF subtypes in which the applied field does not vary with channel height. This paper also discusses the potential role of more powerful microscale field flow fractionation systems as a new class of sample preparation units for micro-total-analysis systems (mu-TAS).     

Sedimentation field-flow-fractionation: emergence of a new cell separation methodology

Field flow fractionation (FFF) methods were conceptualised in the late 1960s by J.C Giddings. These techniques are particularly suited for the retention and separation of micron and sub-micron sized particles. Systematic technological development as well as methodological procedures were established to achieve separations over the last 30 years. The elution mechanism of micron sized species is now known as 'steric/hyperlayer'. Cells are micron sized particles of life science interest, in particular those living in suspension. The separation of cells according to differences in their biophysical characteristics is therefore possible using the FFF principle. In the first part of this report, characteristics of classical cell separation methodologies are recounted as well as the specific features of FFF. In the second part, a review of cell separations or purifications obtained with sedimentation FFF techniques is given and FFF trends in cell separation is developed.     

Separation of carbon nanotubes by frit inlet asymmetrical flow field-flow fractionation

Flow field-flow fractionation (flow FFF), a separation technique for particles and macromolecules, has been used to separate carbon nanotubes (CNT). The carbon nanotube ropes that were purified from a raw carbon nanotube mixture by acidic reflux followed by cross-flow filtration using a hollow fiber module were cut into shorter lengths by sonication under a concentrated acid mixture. The cut carbon nanotubes were separated by using a modified flow FFF channel system, frit inlet asymmetrical flow FFF (FI AFIFFF) channel, which was useful in the continuous flow operation during injection and separation. Carbon nanotubes, before and after the cutting process, were clearly distinguished by their retention profiles. The narrow volume fractions of CNT collected during flow FFF runs were confirmed by field emission scanning electron microscopy and Raman spectroscopy. Experimentally, it was found that retention of carbon nanotubes in flow FFF was dependent on the use of surfactant for CNT dispersion and for the carrier solution in flow FFF. In this work, the use of flow FFF for the size differentiation of carbon nanotubes in the process of preparation or purification was demonstrated.     

Experimental study on the retention of silica particles in gravitational field-flow fractionation effects of the mobile phase composition

Effects of mobile phase composition can play an effective role in modulating the retention of particles in gravitational field-flow fractionation (GFFF), the simplest and cheapest among field-flow fractionation (FFF) techniques. In the framework of an optimized procedure for the GFFF characterization of particulate systems, an experimental approach to the effects of the mobile phase composition on the retention of silica particles retention is presented. The role of the ionic strength and the presence of surfactant are emphasized, with special regards to the shape of the particles. Moreover, the first experimental evidence of potential-barrier GFFF is reported.     

Asymmetrical flow field-flow fractionation technique for separation and characterization of biopolymers and bioparticles

Field-flow fractionation (FFF) is one of the most versatile separation techniques in the field of analytical separation sciences, capable of separating macromolecules in the range 10³–10¹⁵ g mol⁻¹ and/or particles with 1 nm–100 μm in diameter. The most universal and most frequently used FFF technique, flow FFF, includes three types of techniques, namely symmetrical flow FFF, hollow fiber flow FFF, and asymmetrical flow FFF which is most established variant among them. This review provides a brief look at the theoretical background of analyte retention and separation efficiency in FFF, followed by a comprehensive overview of the current status of asymmetrical flow FFF with selected applications in the field of biopolymers and bioparticles.     

Molecular Weight Distributions of Polymer Solutions: Combination of Field Flow Fractionation (FFF) and Analytical Ultracentrifugation (AUC)

Molecular weight, distribution, as well as other molecular characteristics are important drivers in determining the potential behaviors and hence applications of polymeric materials. Out of different methods available for the determination of molecular weight and its distribution, field flow fractionation (FFF) provides absolute molecular weight values and accurate molecular weight distributions. Analytical ultracentrifugation (AUC), on the other hand, relies on the exact density of the polymer materials in solution to determine the accurate molecular weight and its distribution and in the absence of knowledge of exact density, AUC is less accurate than the FFF method. However, combination of the two methods can be achieved to gain insights into the other molecular characteristics of swollen polymer chains. One such example is the determination of the exact density of the swollen polymer chains by the incorporation of the molecular weight information from FFF into AUC analysis. Based on the comparison of the optimized polymer chain density with the bulk density, it was observed that the polyacrylic acid and polyacrylamide chains were swollen in the range of 27 to 29%. Moreover, the FFF and AUC can also complement each other in enhancing the range of characterization possible with the two methods when used separately.     

Osmolarity effects on red blood cell elution in sedimentation field-flow fractionation.

Field-flow fractionation (FFF) is an analytical technique particularly suitable for the separation, isolation, and characterization of macromolecules and micrometer- or submicrometer-sized particles. This chromatographic-like methodology can modulate the retention of micron-sized species according to an elution mode described to date as "steric hyperlayer". In such a model, differences in sample species size, density, or other physical parameters make particle selective elution possible depending on the configuration and the operating conditions of the FFF system. Elution characteristics of micron-sized particles of biological origin, such as cells, can be modified using media and carrier phases of different osmolarities. In these media, a cells average size, density, and shape are modified. Therefore, systematic studies of a single reference cell population, red blood cells (RBCs), are performed with 2 sedimentation FFF systems using either gravity (GrFFF) or a centrifugational field (SdFFF). However, in all cases, normal erythrocyte in isotonic suspension elutes as a single peak when fractionated in these systems. With carrier phases of different osmolarities, FFF elution characteristics of RBCs are modified. Retention modifications are qualitatively consistent with the "steric-hyperlayer" model. Such systematic studies confirm the key role of size, density, and shape in the elution mode of RBCs in sedimentation FFF for living, micronsized biological species. Using polymers as an analogy, the RBC population is described as highly "polydisperse". However, this definition must be reconsidered depending on the parameters under concern, leading to a matricial concept: multipolydispersity. It is observed that multipolydispersity modifications of a given RBC population are qualitatively correlated to the eluted sample band width.     

Improved accuracy in the determination of field-flow fractionation elution volumes.

Conventional operation of field-flow fractionation (FFF) systems involves carrying out the analysis at a constant flow of carrier; the flow is temporarily interrupted after injection of a sample in order to permit its equilibration under the applied field. Retention is calculated as the ratio of elution times for a non-retained species and the sample of interest, respectively. Such time-based retentions are only valid if the flow-rate is precisely known at all times during the run. The peristaltic pumps often used with FFF equipment are shown to have an output which varies unpredictably in time. Furthermore, initiation of flow after relaxation is shown to result in significant periods of transient behaviour while the system adjusts to the operating pressure. These and other variations in flow-rate can be eliminated as sources of error by basing the retention measurement on effluent weight, rather than on time. For this purpose, an electronic balance is interfaced with the system's computer, so that detector response/effluent weight data pairs are continuously monitored during the course of the FFF analysis.     

Field-flow fractionation of proteins, polysaccharides, synthetic polymers, and supramolecular assemblies

This review summarizes developments and applications of flow and thermal field-flow fractionation (FFF) in the areas of macromolecules and supramolecular assemblies. In the past 10 years, the use of these FFF techniques has extended beyond determining diffusion coefficients, hydrodynamic diameters, and molecular weights of standards. Complex samples as diverse as polysaccharides, prion particles, and block copolymers have been characterized and processes such as aggregation, stability, and infectivity have been monitored. The open channel design used in FFF makes it a gentle separation technique for high- and ultrahigh-molecular weight macromolecules, aggregates, and self-assembled complexes. Coupling FFF with other techniques such as multiangle light scattering and MS provides additional invaluable information about conformation, branching, and identity.     

Precision in differential field-flow fractionation: a chemometric study

In the present paper, the capabilities of differential field-flow fractionation, i. e., the determination of an incremental quantity of a colloidal species, e. g., an uptake adsorbed mass, determined by the joint use of two independent FFF measurements, over a species and the same modified species respectively, are considered. The different error types, those related to the retention time determinations and those coming from the operating parameter fluctuations were considered. The different components were computed with reference to SdFFF determinations of bare polystyrene (PS) submicronic particles and the same PS particles covered by IgG. Comparison was made between theoretically computed precision and experiments. The error coming from the experimental measurement of retention times was identified to be the main source of errors. Accordingly, it was possible to make explicit the detection limits and the confidence intervals of the adsorbed mass uptake, as a function of experimental quantities such as the retention ratio, the detector calibration ratio, the injected quantity, the baseline noise, and the void time relative error. An experimentally determined and theoretically foreseen dependence of both the experimental detection and confidence limits (approximately +/- 10(-17) g) on the square root of the injected concentration, for constant injected volume, was found.     

Field-flow fractionation in bioanalysis: A review of recent trends

Field-flow fractionation (FFF) is a mature technique in bioanalysis, and the number of applications to proteins and protein complexes, viruses, derivatized nano- and micronsized beads, sub-cellular units, and whole cell separation is constantly increasing. This can be ascribed to the non-invasivity of FFF when directly applied to biosamples. FFF is carried out in an open-channel structure by a flow stream of a mobile phase of any composition, and it is solely based on the interaction of the analytes with a perpendicularly applied field. For these reasons, fractionation is developed without surface interaction of the analyte with packing or gel media and without using degrading mobile phases. The fractionation device can be also easily sterilized, and analytes can be maintained under a bio-friendly environment. This allows to maintain native conditions of the sample in solution.In this review, FFF principles are briefly described, and some pioneering developments and applications in the bioanalytical field are tabled before detailed report of most recent FFF applications obtained also with the hyphenation of FFF with highly specific, sensitive characterization methods. Special focus is finally given to the emerging use of FFF as a pre-analytical step for mass-based identification and characterization of proteins and protein complexes in proteomics.     

Analysis of complex polymers by multidetector field-flow fractionation

Field-flow fractionation (FFF) is a powerful alternative to column-based polymer fractionation methods such as size-exclusion chromatography (SEC) or interaction chromatography (IC). The most common polymer fractionation method, SEC, has its limitations when polymers with very high molar masses or complex structures must be analysed. Another limitation of all column-based methods is that the samples must be filtered before analysis and shear degradation of large macromolecules may be caused by the stationary phase and/or the column frits. Finally, the separation of very polar polymers may be a challenge because such polymers interact very strongly with the stationary phase, causing irreversible adsorption or other negative effects. This article reviews the latest developments in field-flow fractionation of complex polymers. It is demonstrated that some of the limitations of column-based chromatography can be overcome by FFF. When appropriate, results from column-based fractionations are compared with those from FFF fractionations to highlight the specific merits and challenges of each method. In addition to the fractionations themselves, various detector setups are discussed to show that different polymer distributions require different experimental procedures. Examples are given of the analysis of molar mass distribution, chemical composition, and microstructure. Advanced detector combinations are discussed, most prominently the very recently developed coupling to ¹H NMR. Finally, analysis of polymer nanocomposites by asymmetric flow field-flow fractionation (AF4)–FTIR is presented.

Advances in field-flow fractionation for the analysis of biomolecules: instrument design and hyphenation

Field-flow fractionation (FFF) separates analytes by use of an axial channel-flow and a cross-field. Its soft separation capability makes it an ideal tool for initial fractionation of complex mixtures, but large elution volumes and high flow rates have limited its applicability without significant user handling. Recent advances in instrumentation and miniaturization have successfully reduced channel size and elution speed, and thus the volume of each fraction, making it possible to conveniently couple FFF with orthogonal separation techniques for improved resolution. More detailed analysis can also be performed on the fractions generated by FFF by use of diverse analytical techniques, including MS, NMR, and even X-ray scattering. These developmental trends have given FFF more power in the analysis of different types of molecule, and will be the direction of choice for further advances in FFF technology.     

Fractionation and Size Distribution of Water Soluble Polymers by Flow Field-Flow Fractionation

Abstract

Flow field-flow fractionation (flow FFF) is introduced as a chromatographic-like method with a potential for separating and characterizing water soluble polymers. The theory of the method is summarized, showing that one gets a size distribution curve based on the Stokes diameter, d. Problems in interpreting the elution profile in both flow FFF and gel permeation chromatography are discussed in the light of complications arising from electrostatic chain expansion in polyelectrolytes.

The experimental approach is described using a channel of 2.00 ml volume. Sulfonated polystyrenes of three different molecular weights are separated from one another with and without added salts. The dependence of retention on sample size is shown to be least in the salt solution, indicating that this is most suitable for analytical work.

The sodium salts of polyacrylic acid are also investigated. Distinct elution profiles are noted for two of these polydisperse polymers. Size distribution curves for the 2,000,000 MW sample curves are obtained from, the elution profiles and are shown to be independent of experimental variations. Finally, fractions are collected after separation and rerun through the coloumn, showing a reasonable confirmation of the expected fractionation effect.     

Methods for continuous flow fractionation of microparticles: Outlooks and fields of application

The development of new methods for fractionating particles of a different nature is becoming more important in solving some scientific and technological problems. This paper presents a brief review in the theory and practice of the most common techniques for microparticle fractionation (0.1–100 μm). These are dry and wet sieving, elutriation, sequential filtration, split-flow thin fractionation (SPLITT system), field-flow fractionation (FFF), membrane filtration, and capillary electrophoresis. Special attention is paid to the FFF technique, which offers a unique potential for the separation of different materials, from biopolymers and microorganisms to colloidal and solid particles, and the estimation of their physical properties. An alternative version of sedimentation FFF is described, namely, the fractionation of microparticles in rotating coiled columns. The main advantages and limitations of the methods are revealed and their outlooks and fields of applications are envisaged.     

Determination of particle size distribution by gravitational field-flow fractionation: Dimensional characterization of silica particles

Summary Gravitational field-flow fractionation (GFFF) is the simplest and cheapest of field-flow fractionation (FFF) techniques, although it is still at an early development stage. The application of GFFF to the determination of particle size distribution (PSD) of silica particles used as chromatographic supports is described. The accuracy of the method is evaluated by comparing PSDs obtained by GFFF with those obtained by laser diffraction, a non-separative technique widely applied to particle characterization. It is ultimately demonstrated that a low-cost GFFF channel can simply replace the column of a standard HPLC system, allowing laboratories that are not specialized in size analysis to perform accurate PSD studies with standard HPLC expertise.     

Separation and characterization of nanoparticles in complex food and environmental samples by field-flow fractionation

The thorough analysis of natural nanoparticles (NPs) and engineered NPs involves the sequence of detection, identification, quantification and, if possible, detailed characterization. In a complex or heterogeneous sample, each step of this sequence is an individual challenge, and, given suitable sample preparation, field-flow fractionation (FFF) is one of the most promising techniques to achieve relevant characterization.The objective of this review is to present the current status of FFF as an analytical separation technique for the study of NPs in complex food and environmental samples. FFF has been applied for separation of various types of NP (e.g., organic macromolecules, and carbonaceous or inorganic NPs) in different types of media (e.g., natural waters, soil extracts or food samples).FFF can be coupled to different types of detectors that offer additional information and specificity, and the determination of size-dependent properties typically inaccessible to other techniques. The separation conditions need to be carefully adapted to account for specific particle properties, so quantitative analysis of heterogeneous or complex samples is difficult as soon as matrix constituents in the samples require contradictory separation conditions. The potential of FFF analysis should always be evaluated bearing in mind the impact of the necessary sample preparation, the information that can be retrieved from the chosen detection systems and the influence of the chosen separation conditions on all types of NP in the sample. A holistic methodological approach is preferable to a technique-focused one.     

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). 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.