Different elution modes and field programming in gravitational field-flow fractionation. IV. Field programming achieved with channels of non-constant cross-sections

Force field programming provided increased speed of separation with an improved resolution and detection capability in many field-flow fractionation (FFF) techniques. Gravitational field-flow fractionation (GFFF) uses the Earth's gravitational field to cause the settlement of particles towards the channel accumulation wall. Although this field is constant and relatively weak, there are different ways to implement force field programming in GFFF. Because hydrodynamic lift forces (HLF) participate in the separation process in focusing (hyperlayer) elution mode, they can control the resulting force field acting on particles via changes in flow-velocity. These changes can be accomplished by a programmable pump or with channels of non-constant cross-sections. This work is focused on flow-velocity programming accomplished with channels of non-constant cross-sections. Three trapezoidal channels of decreasing breadth and two channels of decreasing height (along the longitudinal axis) are tested as tools for optimization of the separation of a model silica gel particle mixture. The trapezoidal channels yielded reduced separation times. However, taking into account both speed of separation and resolution, the optimization effect was lower compared with the flow-rate gradients generated by a programmable pump. The channels of non-constant height did not yield advantageous separations.     

Hyperlayer separation in hollow fiber flow field-flow fractionation: effect of membrane materials on resolution and selectivity

Hollow fiber flow FFF (HF FlFFF) has recently shown its capability to separate and characterize the size of submicrometer particles and has demonstrated the potential to be developed into a disposable flow FFF channel. In this work, HF FlFFF was used for the hyperlayer separation of micron-sized particles and the separation capability was examined by using various hollow fiber membrane materials (Polysulfones, cPVC, and PAN). From the experiments, PAN (polyacrylonitriles) showed an outstanding performance in particle separation compared to the other membranes. By orienting the fiber module in an upright direction, the upstream flow migration reduced band broadening of eluted peaks. When the efficiency of the PAN hollow fiber system was tested by varying the ratio of outflow-rate to radial flow-rate, it was found that optimum separation in hyperlayer HF FlFFF can be obtained at the ratio of about 6–7. From the examination of retention at or around steric inversion diameter, it was observed that experiments showed a good agreement with predictions by semi-empirical calculation. In hyperlayer HF FlFFF the diameter based selectivity values were shown to be 1.2–1.7 depending on the type of membranes and the field strength (the radial flow-rate) conditions.     

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。     

Computer simulation of particle separation based on non-equilibrium swelling

Steric/hyperlayer field-flow fractionation (FFF) is an established analytical technique for separating and characterizing particles in the 1-100 microns diameter range. The separation can be based on differences in size, density, shape and mechanical properties of the particles. In the course of an analysis of the water transporter system of Chinese hamster ovary (CHO) cells and one of their high permeability mutants, the first successful attempt was made to use the steric/hyperlayer FFF system for the purpose of separating particles based on a time-dependent property, namely, the differential swelling of the two cell types. The present study was undertaken to simulate numerically the separation and suggest selection of operating conditions to minimize repetitive experiments. The computer simulation was developed using Maple V, a symbolic computing environment. It is shown that the model is able to predict an optimal velocity of carrier buffer that maximizes resolution. Predicted velocity/resolution pairs are in good agreement with available experimental data. Empirical models for the lift forces encountered in such FFF experiments, and for the zone broadening observed in work with cell sized particles, form the basis for this model.     

Different elution modes and field programming in gravitational field-flow fractionation. III. Field programming by flow-rate gradient generated by a programmable pump.

Gravitational field-flow fractionation (GFFF) utilizes the Earth's gravitational field as an external force that causes the settlement of particles towards the channel accumulation wall. Hydrodynamic lift forces oppose this action by elevating particles away from the channel accumulation wall. These two counteracting forces enable modulation of the resulting force field acting on particles in GFFF. In this work, force-field programming based on modulating the magnitude of hydrodynamic lift forces was implemented via changes of flow-rate, which was accomplished by a programmable pump. Several flow-rate gradients (step gradients, linear gradients, parabolic, and combined gradients) were tested and evaluated as tools for optimization of the separation of a silica gel particle mixture. The influence of increasing amount of sample injected on the peak resolution under flow-rate gradient conditions was also investigated. This is the first time that flow-rate gradients have been implemented for programming of the resulting force field acting on particles in GFFF.     

Optimization of cyclical electrical field flow fractionation

Cyclical electrical field flow fractionation (CyElFFF) is a variation on electrical field flow fractionation (ElFFF) where cyclical electrical fields are used instead of steady DC fields to increase the effective field experienced by particles in the flow channel. Even though the effective field increases more than 20-fold compared to normal ElFFF, the retention and resolution in CyElFFF has not been shown to be better than in ElFFF. In this paper we report how one can optimize operational parameters in CyElFFF to obtain good retention and resolution in CyElFFF. The effects of offset voltage, frequency, flowrate, concentration of particles and sample size on retention, resolution and retained peak/void peak ratio have been observed. The results obtained from these experiments were analyzed and suggestions have been made to improve both retention and resolution. A 4-fold improvement in retention without a significant increase in band broadening is reported.     

重力场流分离系统用于聚苯乙烯颗粒的分离条件优化

重力场流分离是最简单的场流分离(gravitational flow-field fractionation,GrFFF)技术,常用于分离粒径几微米到几十微米的颗粒及生物样品。利用自组装加工的重力场流分离仪器分离3种不同粒径(3、6、20μm)的聚苯乙烯(PS)颗粒。自制了一种混合表面活性剂,并与商品化的表面活性剂FL-70进行了比较。通过均匀设计优化流速、混合表面活性剂中聚乙二醇辛基苯基醚(Triton X-100)的质量分数、载液黏度、停流时间等分离条件,以分离度(Rs)和保留比(R)为评价指标,发现FL-70的分离效能略优于自制的混合表面活性剂,可实现3种PS颗粒的完全分离(Rs1为1.771,Rs2为2.074)。结果表明该系统具有良好的分离性能。     

Low-capacity channel designed for particle separation with controlled electric fields and evaluation of involved forces

Electric field is one of the suitable physical fields applicable to particle separations. Although long rectangular channel is used for particle separation in usual electrical field flow fractionation (FFF), a short low-capacity channel can replace it if the field is precisely controlled. Several separation principles are proposed with this channel. The elution behavior of particles has revealed that the gravitational, diffusion, and hydrodynamic lift force (HLF) play important roles in the determination of the elution behavior of particles. The elution threshold voltage (V(th)) was defined and experimentally determined for various system configurations and particles. The electric force no longer overcomes the other forces, and particles are taken off the wall, when the applied voltage becomes lower than V(th). V(th) values have allowed us not only to estimate surface charge density of a particle but also to evaluate the hydrodynamic lift force against particle.     

Different elution modes and field programming in gravitational field-flow fractionation: field programming using density and viscosity gradients

In previous papers, several approaches to programming of the resulting force field in GFFF were described and investigated. The experiments were dealing with flow-velocity and channel thickness, i.e. factors influencing hydrodynamic lift forces (HLF). The potential of density and viscosity of carrier liquid for field programming was predicted and demonstrated by preliminary experiments. This work is devoted to experimental verification of the influence of carrier liquid density and viscosity. Several carrier liquid density and simultaneously viscosity gradients using water-methanol mixtures are in this work implemented in the separation of a model silica mixture. Working with the water-methanol gradients, one is not able to separate the influence of density from the contribution of viscosity. However, we found experimental conditions to show the isolated effect of carrier liquid density (two water-methanol mixtures of equal viscosity differing in their densities). In order to demonstrate the isolated effect of viscosity, we implemented in this work a new system of (hydroxypropyl)methyl cellulose (HPMC) carrier liquids. Three different HPMC compositions enabled to vary the viscosity more than two times at almost constant density. With increasing carrier liquid viscosity, the focusing and elevating trend was clearly pronounced for 5 and 10 microm silica particles. By the isolated effect of increased viscosity, the centre of the 10 microm particle zone was elevated to the streamline at 16% of the channel height. These experiments have shown that the influence of carrier liquid viscosity on HLF should be taken into account even at higher levels above the channel bottom, i.e. beyond the near-wall region. Further, it is shown that higher value of carrier liquid viscosity improves the separation of the model mixture in terms of time and resolution.     

Hyperlayer hollow-fiber flow field-flow fractionation of cells

Interest in low-cost, analytical-scale, highly efficient and sensitive separation methods for cells, among which bacteria, is increasing. Particle separation in hollow-fiber flow field-flow fractionation (HF FlFFF) has been recently improved by the optimization of the HF FIFFF channel design. The intrinsic simplicity and low cost of this HF FlFFF channel allows for its disposable usage. which is particularly appealing for analytical bio-applications. Here, for the first time, we present a feasibility study on high-performance, hyperlayer HF FIFFF of micrometer-sized bacteria (Escherichia coli) and of different types of cells (human red blood cells, wine-making yeast from Saccharomyces cerevisiae). Fractionation performance is shown to be at least comparable to that obtained with conventional, flat-channel hyperlayer FIFFF of cells, at superior size-based selectivity and reduced analysis time.     

Different elution modes and field programming in gravitational field-flow fractionation. 2. Experimental verification of the range of conditions for flow-rate and carrier liquid density programming

Gravitational field-flow fractionation utilises the Earth's gravitational field as an external force that causes the settlement of particles towards the channel accumulation wall. Hydrodynamic lift forces oppose this action by elevating of particles from the channel accumulation wall. Therefore there are several possibilities to modulate the resulting force field acting on particles in gravitational field-flow fractionation. Regarding the force field programming in gravitational field-flow fractionation, this work focused on two topics: changes of the difference between particle density and carrier liquid density in Brownian and focusing elution modes and influencing of lift forces achieved by changing the flow-rate in focusing elution mode. We have found and described the experimental conditions applicable to force field programming in the case of separations of silica gel particles by gravitational field-flow fractionation. It was shown that the effect of carrier liquid viscosity in the water-methanol system is implemented as an additional factor enhancing the desired effect of carrier liquid density. Some other forces influencing the retention behaviour of the model particles are discussed.     

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.     

Elimination of edge effects in micro-thermal field-flow fractionation channel of low aspect ratio by splitting the carrier liquid flow into the main central stream and the thin stream layers at the side channel walls

An optimized construction of the separation channel for micro-thermal field-flow fractionation (FFF) was proposed and studied experimentally. The sample is injected in such a manner that its zone moves along the channel only in the main central stream where the flow velocity profile in the plane parallel to the main accumulation wall is practically flat. This central stream is separated from the contact with the side walls of the channel by thin flowing layers of the free carrier liquid. The retained species do not reach the thin liquid streams at the side walls where the flow rate decreases rapidly to achieve zero at the side wall according to the established 3D flow velocity profile. Such a construction of the channel allows one to reduce the aspect ratio (the ratio of the channel breadth b to its thickness w) without increasing the zone broadening. The hydrodynamic splitting of the outlet streams allows one not only to increase the concentration of the detected species but also the determination of the sign of Soret coefficient.     

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.     

Biocompatible channels for field-flow fractionation of biological samples: correlation between surface composition and operating performance

Biocompatible methods capable of rapid purification and fractionation of analytes from complex natural matrices are increasingly in demand, particularly at the forefront of biotechnological applications. Field-flow fractionation is a separation technique suitable for nano-sized and micro-sized analytes among which bioanalytes are an important family. The objective of this preliminary study is to start a more general approach to field-flow fractionation for bio-samples by investigation of the correlation between channel surface composition and biosample adhesion. For the first time we report on the use of X-ray photoelectron spectroscopy (XPS) to study the surface properties of channels of known performance. By XPS, a polar hydrophobic environment was found on PVC material commonly used as accumulation wall in gravitational field-flow fractionation (GrFFF), which explains the low recovery obtained when GrFFF was used to fractionate a biological sample such as Staphylococcus aureus. An increase in separation performance was obtained first by conditioning the accumulation wall with bovine serum albumin and then by using the ion-beam sputtering technique to cover the GrFFF channel surface with a controlled inert film. XPS analysis was also employed to determine the composition of membranes used in hollow-fiber flow field-flow fractionation (HF FlFFF). The results obtained revealed homogeneous composition along the HF FlFFF channel both before and after its use for fractionation of an intact protein such as ferritin.     

Particle size analyses of porous silica and hybrid silica chromatographic support particles. Comparison of flow/hyperlayer field-flow fractionation with scanning electron microscopy, electrical sensing zone, and static light scattering

Porous silica and hybrid silica chromatographic support particles having particle diameters ranging approximately from 1 microm to 15 microm have been characterized by flow/hyperlayer field-flow fractionation (FFF). The particle size accuracy has been improved significantly in this work by a second-order polynomial calibration. Very good agreement between the FFF data and scanning electron microscopic (SEM) results has been achieved. The effects of particle porosity, pore sizes, and particle sizes on the particle size accuracy in electrical sensing zone (ESZ) analyses have been discussed. It has been demonstrated by computer simulation and experimental measurements that false peaks can be generated in certain particle size regions when the static light scattering (SLS) technique is applied to tightly distributed spherical chromatographic support particles.     

Inertial separation in a contraction-expansion array microchannel

We report a contraction-expansion array (CEA) microchannel that allows inertial size separation by a force balance between inertial lift and Dean drag forces in fluid regimes in which inertial fluid effects become significant. An abrupt change of the cross-sectional area of the channel curves fluid streams and produces a similar effect compared to Dean flows in a curved microchannel of constant cross-section, thereby inducing Dean drag forces acting on particles. In addition, the particles are influenced by inertial lift forces throughout the contraction regions. These two forces act in opposite directions each other throughout the CEA microchannel, and their force balancing determines whether the particles cross the channel, following Dean flows. Here we describe the physics and design of the CEA microfluidic device, and demonstrate complete separation of microparticles (polystyrene beads of 4 and 10 μm in diameter) and efficient exchange of the carrier medium while retaining 10 μm beads.     

Size selectivity in field-flow fractionation: lift mode of retention with near-wall lift force

A simple theoretical model for the size selectivity, S(d), in the lift mode of retention in field-flow fractionation (FFF) is developed on the basis of the near-wall lift force expression. S(d) is made up of two contributions: the flow contribution, S(d,f), arising from the variation of the flow velocity at center of particle due to a change in particle position with particle size, and a slip contribution, S(d,s), arising from the concomitant change in the extent of retardation due to the presence of a nearby channel wall. The slip contribution is minor, but not negligible, and amounts to 10-20% of the overall size selectivity. It contributes to reduce S(d) in sedimentation FFF but to enhance it in flow FFF. S(d) would steadily increase with particle size if the flow profile was linear (Couette flow). Because of the curvature of the flow profile encountered in the classical Poiseuille flow, S(d) exhibits a maximum at some specific particle size. The model predicts a significant difference in S(d) between sedimentation FFF and flow FFF, arising from the different functional dependences of the field force with particle size between these two methods. The predictions are in good agreement with the various S(d) values reported in the literature in both sedimentation and flow FFF. On the basis of the model, guidelines are given for adjusting the operating parameters (carrier flow rate and field strength) to optimize the size selectivity. Finally, it is found that S(d) generally decreases with decreasing channel thickness.     

Gravitational field-flow fractionation in combination with flow injection analysis or electrothermal AAS for size based iron speciation of particles

A simple gravitational field-flow fractionation (GrFFF) system was used for size separation of micron sized silica particles coated with hydrous iron oxide (geothite). The amount of iron on the particles was monitored either on-line by reverse-flow injection analysis (r-FIA) with chemiluminescence detection using luminol or off-line by electrothermal atomic absorption spectrophotometry (ETAAS). The combination of GrFFF with reverse FIA or with ETAAS has been demonstrated to be a cost-effective tool for size based iron speciation of particles.     

Gravitational field-flow fractionation for the characterisation of active dry wine yeast.

Gravitational field-flow fractionation (GrFFF) is applied to the fractionation of active dry wine yeast. An experimental approach to the analysis of the effects that field variation by changing mobile phase composition and flow-rate have on the fractionation process of standard particles (polystyrene) was first developed to further obtain effective fractionation of wine yeast by GrFFF. Scanning electron microscopy and Coulter counter particle size measurements were used to monitor the fractionation extent and capabilities of GrFFF to describe the distribution of yeast cells populations.