Increased size‐sorting performance in gravitational SPLITT by using a pinched sample inlet design

Split‐flow thin fractionation is a continuous, flow‐assisted separation technique for sorting macromolecules and particulate matter on a preparative scale. On reducing the thickness of the sample inlet conduit of a gravitational split‐flow thin fractionation channel, size‐sorting performance is found to increase since particles that are continuously fed into the channel can be more rapidly compressed toward the upper wall of the channel. Experiments are carried out by measuring the number percentage of particles eluted at each outlet as a function of different thickness values of the sample inlet conduit. The effects that the total thickness of the gravitational split‐flow thin fractionation channel and the sample feed concentration have on the size‐fractionation performance are examined with the goal of determining the best pinched sample inlet, gravitational split‐flow thin fractionation channel design.     

Membrane filtration for the continuous fractionation of species and macromolecules: Component distribution in the Arzhaan spring water

An improved device was proposed for the fractionation and preconcentration of particles and macromolecules. The device provides new means for the separation of water components. In the proposed method, a tangential flow of the test solution is passed through a series of successive membranes with decreasing pore size. The method was applied to the fractionation of low-mineralized water (Ty va Arzhaanes) with low concentrations of trace components.     

Size Characterization of Pharmaceutical Microspheres by Flow Field-Flow Fractionation

Flow field flow fractionation (FIFFF), one of the subtechniques in FFF family, is a separation technique that can be applied for the separation and characterization of particulate materials, biological macromolecules, and water soluble polymers. Separation in FIFFF is carried out in an empty channel by the interaction of applied field from an external source with flow. Retention of particles or macromolecules in FIFFF is governed by the relative protrusion of sample materials to the differential flow streamlines. Thus in FIFFF, particle size can be readily calculated from the experimental fractogram by theory or calibration.     

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.     

Field and flow programming in frit-inlet asymmetrical flow field-flow fractionation

The separation of wide molecular mass (Mr) ranges of macromolecules using frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF) has been improved by implementing a combination of field and flow programming. In this first implementation, field strength (governed by the cross flow-rate through the membrane-covered accumulation wall) is decreased with time to obtain faster elution and improved detection of the more strongly retained (high Mr) materials. The channel outlet flow-rate is optionally held constant, increased, or decreased with time. With circulation of the flow exiting the accumulation wall to the inlet frit, the dual programming of cross flow and channel outlet flow could be implemented using just two pumps. With this flow configuration, the channel outlet flow-rate is always equal to the channel inlet flow-rate, and these may be programmed independently of the cross flow-rate through the membrane. FI-AFlFFF retains its operational advantage over conventional asymmetrical flow FFF (AFlFFF). Unlike conventional AFlFFF, FI-AFlFFF does not require time consuming, and experimentally inconvenient, sample focusing and relaxation steps involving valve switching and interruption of sample migration. The advantages of employing dual programming with FI-AFlFFF are demonstrated for sets of polystyrene sulfonate standards in the molecular mass range of 4 to 1000 kDa. It is shown that programmed FI-AFlFFF successfully expands the dynamic separation range of molecular mass.     

Hybrid gravitational field-flow fractionation using immunofunctionalized walls for integrated bioanalytical devices

In this work, the biospecific recognition antigen–antibody reaction was implemented in gravitational field-flow fractionation (GrFFF), a flow-assisted separation technique for micron-sized particles, in order to realize a hybrid immunomodulated GrFFF system in which two different principles are combined to achieve highly versatile fractionation. Micron-sized polystyrene beads coated with horseradish peroxidase (HRP) were used as a model sample, and anti-HRP antibodies were immobilized on the accumulation wall of the GrFFF channel. Ultrasensitive chemiluminescence imaging was employed to visualize the beads during elution and to optimize experimental conditions. The same principle was then applied to real biological samples composed by Yersinia enterocolitica and Escherichia coli cells. Results show the possibility to modify the elution of selected sample components and even to retain them into the channel. The hybrid immunomodulated GrFFF system is a step towards the development of a module that could be integrated in a lab-on-a-chip-based point-of-care testing device which includes sample pre-analytical cleanup and analysis.     

重力场流分离系统中载液及流速条件对分离的影响

重力场流分离作为最简单的一种场流分离技术,常用于分离微米级颗粒。选择两种不同粒径(20 μ m和6 μ m)的聚苯乙烯(PS)颗粒作为样品,通过改变载液中叠氮化钠浓度、混合表面活性剂的比例及载液流速,利用自行设计生产的重力场流分离(gravitational flow field-flow fractionation, GrFFF)仪器,对颗粒混合样品进行分离,得到了相关谱图与数据,考察了这3种因素对分离效果(保留比(R)、塔板高度(H))的影响。结果表明:20 μ m PS颗粒的R值均大于6 μ m PS颗粒的R值,H值均小于6 μ m颗粒的H值;PS颗粒的R值与H值均随着载液中叠氮化钠浓度的增加而增加;但随着载液流速的增加,R值增加,H值减小。该研究为GrFFF系统的开发及应用提供了重要的参考价值。     

Performance of hollow-fiber flow field-flow fractionation in protein separation

Since hollow-fiber flow field-flow fractionation (HF FIFFF) utilizes a cylindrical channel made of a hollow-fiber membrane, which is inexpensive and simple in channel assembly and thus disposable, interests are increasing as a potential separation device in cells, proteins, and macromolecules. In this study, performance of HF FIFFF of proteins is described by examining the influence of flow rate conditions and length of fiber (polyacrylonitrile or PAN in this work) on sample recovery as well as experimental plate heights. The interfiber reproducibility in terms of separation time and recovery was also studied. Experiments showed that sample recovery was consistent regardless of the length of fiber when the effective field strength (equivalent to the mean flow velocity at the fiber wall) and the channel void time were adjusted to be equivalent for channels of various fiber lengths. This supported that the majority of sample loss in HF FIFFF separation of apoferritin and their aggregates may occur before the migration process. It is finally demonstrated that HF FIFFF can be applied for characterizing the reduction in Stokes' size of low density lipoproteins from blood plasma samples obtained from patients having coronary artery disease and from healthy donors.     

Evaluation of multiplexed hollow fiber flow field-flow fractionation for semi-preparative purposes

A multiplexed hollow fiber flow field-flow fractionation (MxHF5) is introduced to increase throughput of an HF5 channel system for semi-preparative purposes. HF5, a cylindrical version of the flow field-flow fractionation (FlFFF) operated with a porous, hollow fiber membrane by controlling the ratio of radial and axial flow rates, is capable of fractionating proteins, cells, and macromolecules by size. An advantage of HF5 is its inexpensive channel construction, allowing for disposability that can reduce run-to-run carryover problems. MxHF5 constructed in this study was made with six parallel HF5 modules connected to seven-port manifolds for the semi-preparative scale separation of proteins or biological particles. For the evaluation of MxHF5 separation efficiency, protein standards were utilized to test peak recoveries, band broadening, and throughput. The assembly showed the possibility of handling up to 50 μg of proteins without incurring overloading. The developed channel was applied to demonstrate size sorting of lipoproteins for the future study of size dependent lipidomic and proteomic analysis. The current trial offers a unique advantage of scaling up HF5 separation without using wide-bore, hollow fibers which sacrifice separation speed.     

Improvement of lipoprotein separation with a guard channel prior to asymmetrical flow field-flow fractionation using fluorescence detection

In this article, a simple experimental approach to improve lipoprotein separation and detection in flow field-flow fractionation (FlFFF) is detailed. Lipoproteins are globular particles composed of lipids and proteins in blood serum and their roles include transferring fats and cholesterols through blood vessels throughout the body. Especially, presence of small, dense low-density lipoproteins (LDL) is associated with cardiovascular risk. Two experimental approaches were explored in this study: an increase in the reproducibility of LDL particle separation by implementing a guard channel prior to an asymmetrical FlFFF (AFlFFF) channel in order to deplete small molecular weight serum proteins and reducing the required injection volume of a serum sample by implementing fluorescence detection. The guard channel was made of a simple hollow fiber module so that the serum sample can be washed with the help of radial flow prior to injection into the AFlFFF channel. The channel was tested with protein standards and serum samples to ensure precision of the retention time and the protein recovery rate. A fluorescent phospholipid dye was utilized to label lipoprotein particles before separation for fluorescence detection, which resulted in a reduction of the required injection volume of serum.     

Preparative continuous flow electrophoretic instrumentation for purification of biological samples

We constructed a preparative instrumentation and developed the methods that are based on separation of the samples by bidirectional isotachophoresis/moving boundary electrophoresis in continuous divergent flow. The described instrumentation can be used for a variety of the samples, however, it can be easily optimized and tailored for the specific sample. The trapezoid separation bed from nonwoven textile exhibited minimum adsorption effect for sample and it can be used repeatedly. By the addition of different spacers via separation space inlets, the sections of pH gradient can be modified to enhance the separation. The liquid flow from two inlets positioned on each side of the sample inlet prevented the contact of the sample with anolyte and catholyte at the analysis beginning. One pair of thin electrodes (graphite and stainless-steel) was placed at the separation space output. The electrode products were washed out into drains without disturbing the focusing process. The influence of EOF was managed by tilting the separation bed in the direction from cathodic to anodic side. The components of spirulina supernatant and color pI markers were separated in the pH gradient from 3.9 to 10.1. pH gradient was stable for at least 4.5 h and spirulina supernatant from about 0.12 g of dry powder was processed. Compared to other preparative methods used for spirulina separation, the presented method/instrumentation working with a continuous divergent flow had essential advantages. The efficient separation was fast, and no intermediate steps were necessary to obtain liquid fractions with separated components compatible with further biological experiments.     

Micro-thermal field-flow fractionation: new high-performance method for particle size distribution analysis

Micro-thermal field-flow fractionation (mu-TFFF) was applied to the separation of polystyrene latices. This new high-resolution technique allows determination of the particle size distribution (PSD) if carried out under optimized experimental conditions. The optimum temperature of the accumulation wall, which influences the relaxation processes and, consequently, the zone broadening, was chosen on the basis of our prior work. The flow rate was chosen as a compromise between the theoretical optimum value, which is very low because the diffusion coefficients of the colloidal particles are very small, and a value allowing performance of the PSD analysis in a reasonable time. These experimental conditions can be manipulated easily due to the high versatility of mu-TFFF, which follows from a large decrease of the heat energy flux across the channel with its reduced dimensions in comparison with standard TFFF. The PSDs obtained from mu-TFFF data are compared with results from quasi-elastic laser light scattering (QELS) and transmission electron microscopy (TEM). It has been found that a baseline resolution of a model mixture of two samples of close average particle diameters can be achieved by an appropriate choice of the temperature drop in mu-TFFF, whereas only a broad, unresolved PSD of the mixed sample was obtained from the QELS measurement. The TEM of the mixed sample revealed the presence of two particle size populations. However, the number of particles which are practically counted on a TEM picture is several orders of magnitude lower than the number of particles taken into account in mu-TFFF or QELS. Consequently, the PSD obtained from the TEM did not represent the whole sample. Comparison of mu-TFFF with modern hydrodynamic chromatography (HC) has shown that the methods exhibit roughly the same resolution and time of analysis. Nevertheless, mu-TFFF is a more universal technique because the separation of the colloidal particles or of the macromolecules within a broad range of molar masses is carried out on the same channel, as demonstrated previously.     

Separation and characterization of particles in natural water and soil using flow field-flow fractionation

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

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

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

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.     

The problem of bimodal distributions in dynamic light scattering: Theory and experiment

A new method is proposed for analyzing the results of dynamic light scattering measurements of polymer solutions containing macromolecules with substantially different molecular masses. The processing algorithm makes it possible to correctly allow for effects of the quality of solvent and the conformation and polarizability of macromolecules on the contributions of components of a system to the intensity of scattering. The developed method is tested for a model mixture of two polystyrene samples dissolved in a good solvent (tetrahydrofurane) and a θ solvent (cyclohexane) and is applied to study a system containing a linear polymer and an interpolymer complex.     

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

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

Phase-distribution chromatography (PDC) of polystyrene

A chromatographic technique is described where the stationary phase is a layer of a very high molecular polystyrene fraction (M = 10⁷) on glass beads (ballotines). The mobile phase is cyclohexane passing the column at a constant temperature below the theta-temperature. A polystyrene sample of sufficiently low molecular weight (M ≤ 10⁶) injected as a small plug at the top of the column is fractionated because the distribution between the mobile and the stationary phase depends on the molecular weight. Since the large molecules preferentially remain in the stationary phase, the smaller molecules leave the column first. The fractionation effect is inverse to that found in GPC experiments. The separation efficiency is rather good and can be described by a simple thermodynamic theory.     

Flow field-flow fractionation: recent trends in protein analysis

Flow field-flow fractionation (F4) is the gentlest flow-assisted separation technique for analysis of macromolecules. The use of an empty channel as separation device and of a second mobile phase flow as perpendicular field enable F4 to separate analytes under native conditions without any modification of their original structure. Because of this unique peculiarity, F4 has been shown to be ideal for "gentle" separation of biological samples, for example intact proteins and protein complexes, since its early development. Today's F4 is an appealing technique which complements most established separation techniques, for example liquid chromatography and electrophoresis. The number of applications that show the unique advantages of F4 for analysis of protein samples is constantly increasing. In particular, F4 is finding increasing application on very high-molecular-weight species such as protein oligomers, aggregates, and complexes. This review critically discusses recent literature on the application of F4 to proteins. Either stand-alone or coupled with other characterization techniques, F4 is particularly promising for quality control of protein therapeutics, characterization of amyloid proteins, lipoprotein profiling, and as a pre-MS separation step in proteomics.     

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

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