Hydrodynamic relaxation in flow field-flow fractionation using both split and frit inlets.

Two means are described for achieving hydrodynamic relaxation and thus avoiding the stopflow injection procedure in field-flow fractionation (FFF): split flow injection and frit inlet injection. The advantages, disadvantages, and the theoretical basis of these procedures are discussed. Incremental band broadening due to the final relaxation step is examined theoretically and shown to be negligible when the flow rate of the sample inlet substream is small compared to the total channel flow rate. The optimization of the sample inlet flow rate is discussed. Experimental results for both injection procedures are reported for flow/steric (or hyperlayer) FFF applied to latex standards, confirming the expected trends. However, closer examination shows that the observed incremental band broadening associated with hydrodynamic relaxation is somewhat larger than the value predicted.     

Hydrodynamic relaxation and sample concentration in field-flow fractionation using permeable wall elements

The advantages of hydrodynamic relaxation in field-flow fractionation, in which an injected sample is driven rapidly toward its equilibrium distribution by flow, are described relative to conventional field-driven relaxation. A new concept for achieving hydrodynamic relaxation, based on the use of permeable wall elements (or frit elements) embedded in the channel walls, is introduced. Here an auxiliary substream of carrier fluid, permeating uniformly into the FFF channel near the inlet, drives the sample, entrained in its own substream, close to its equilibrium configuration. Such frit elements can also be used to enrich the sample at the outlet. Equations are derived and plots are provided for the position of the splitting plane dividing the two substreams; this position defines the strength of the hydrodynamic relaxation. Variations in shear through these frit-modified end regions are also formulated and plotted. The effects of frit elements on band broadening are discussed. It is concluded that permeable wall elements in many configurations may be broadly applicable to FFF and related methods for improved sample introduction, increased separation speed, reduced risk of sample adhesion to the wall, improved flow stability, and sample enrichment.     

Miniaturization of frit inlet asymmetrical flow field-flow fractionation

A miniaturized frit inlet asymmetrical flow field-flow fractionation (mFI-AFlFFF) channel has been constructed and tested for the separation of proteins. By scaling down the geometrical channel dimension of a conventional FI-AFlFFF system, flow rate ranges that can be manipulated were decreased to 20-30 microL/min, which reduces the injection amount of sample materials. The end effect contribution to plate height was evaluated by varying the inner diameter of the connection tubing between the injector and the channel inlet at various injection flow rates, and the results showed that the use of silica capillary tubing of the shortest possible distance is essential in reducing the initial band broadening prior to the sample injection to the microscale channel. The capability of the microFI-AFlFFF system was demonstrated with the separation of protein standards, polystyrenesulfonates, and ssDNA strains and for the characterization of replication protein A-ssDNA binding complex regulated by redox status.     

Influence of operating parameters on the retention of chromatographic particles by thermal field-flow fractionation

We have investigated the retention behavior of chromatographic particles in thermal field-flow fractionation (FFF). Retention time is found to increase with increasing temperature drop across the channel thickness, as expected for species exhibiting a thermophoretic mobility. Experiments have been performed with a vertically oriented channel rather than by using the classical horizontal configuration as this leads to much more reproducible retention data. In acetonitrile, silica-based particles are more retained than octadecyl-bonded silica particles, which confirms our previous finding, by means of a different method, that the thermophoretic mobility of the latter is smaller than that of the former. Whatever the type of particles and the nature of the carrier liquid, the relative retention time is observed to decrease with increasing carrier flow rate. This indicates that a hydrodynamic lift force acts on particles to move them away from the accumulation wall, as is usually observed in all FFF experiments with micrometer-sized particles. However, upward and downward flow directions in the vertical channel lead to similar retention data, indicating that inertial lift forces have a minor influence on retention. In addition, the relative retention time steadily decreases with increasing sample concentration, suggesting that the hydrodynamic lift force increases significantly with sample concentration. Accordingly, we speculate that a new transport phenomenon, called shear-induced hydrodynamic diffusion, not previously accounted for in the modeling of retention in FFF, is controlling the migration of the particles in the FFF channel. Implications of the influence of this phenomenon in other FFF experiments are discussed.     

Stopless flow injection in asymmetrical flow field-flow fractionation using a frit inlet

Stopless flow operation of asymmetrical flow field-flow fractionation (FFF) has been achieved by introducing a hydrodynamic relaxation method using a frit inlet. By using frit inlet injection, a focusing process which has been an essential part of runs at the asymmetrical flow FFF system can be completely avoided. Band broadening of an initial sample zone during hydrodynamic relaxation is discussed with equations related to the ratio of two inlet flow rates. For the successful achievement of particle relaxation and separation, it is necessary to apply a small ratio of sample inlet to frit inlet flow rate. Experimental results are reported for the evaluation of the system efficiency at various levels of hydrodynamic relaxation and for both normal and steric/hyperlayer modes of FFF runs using latex standards. Most importantly, it is shown that a high resolution and a high-speed separation of submicrometer-sized latex mixtures can be accomplished in asymmetrical flow FFF without using the conventional focusing relaxation process.     

Working without accumulation membrane in flow field-flow fractionation

Nonideal interaction of sample with the separation device is a difficulty found in chromatographic methods as well as in field-flow fractionation. However, in field-flow fractionation (FFF), greater flexibility in the choice of carrier solution composition is possible, thus reducing the need of a wide choice of surface chemistry when nonideal sample interaction is to be minimized. The use of an ultrafiltration membrane as the surface for the accumulation wall is common practice in flow field-flow fractionation. Typical membranes in use are laminates of a skin membrane onto a backing material such as woven polyester. At this point, only a limited choice of membrane chemistries is available. Many membranes have been developed for protein applications as membranes are widely used in the pharmaceutical industries. While these membranes work well for protein applications, flow field-flow fractionation is applicable to polymeric particulate as well as protein samples. Thus, sample interaction with the membrane surface is possible with nonprotein applications and these interactions can induce significant secondary effects on retention ratio and affect instrumental reliability. Also, the woven texture of membranes may detrimentally affect the FFF separation. For these reasons, the study of flow field-flow fractionation using a flat, smooth surface of controlled chemistry is of relevance. We present here the results of a new, membraneless channel that uses a bare frit as the accumulation wall and that is intended for analysis of micrometer-sized particles only. Selectivity results are comparable to those obtained with the membrane, while relative sample recovery indicates that the best quantitative performance can be obtained without the membrane. Moreover, neither sample immobilization nor losses through the frit occur when operating membraneless. On the other hand, first experimental evidence of a certain level of frit surface activity suggests that optimization of experimental conditions is required.     

Determination of mean diameter and particle size distribution of acrylate latex using flow field-flow fractionation, photon correlation spectroscopy, and electron microscopy

Flow field-flow fractionation (flow FFF) was employed to determine the mean diameter and the size distribution of acrylate latex materials having diameters ranging from 0.05 to 1 μm. Mean diameters of the samples determined by flow FFF are in good agreement with those obtained from photon correlation spectroscopy (PCS). Scanning electron microscopy (SEM) yielded a mean diameter that is about 20% lower than those obtained from flow FFF or PCS, probably due to the shrinkage of particles during sample drying and high-vacuum measurements. It was found that flow FFF is particularly useful for the determination of particle size distributions of latex materials having broad size distributions. Flow FFF separates particles according to their sizes and yields an elution curve that directly represents the particle size distribution of the sample. In PCS, measurements had to be repeated at more than one scattering angle to obtain an accurate mean diameter for the latex having a broad size distribution. Flow FFF was fast (less than 12 min of run time) and showed an excellent repeatability in measuring the mean diameter with ±5% relative error.     

Circular asymmetrical flow field-flow fractionation for the semipreparative separation of particles

A new technique for the separation and characterization of particles and polymers based on asymmetrical flow field-flow fractionation was developed. The new circular asymmetrical flow field-flow fractionation instrument (CAFFFE) resembles a quasi-parallel arrangement of 12 individual flow channels. As compared to the classical asymmetrical flow field-flow fractionation (AF-FFF), which can be used so far only for analytical separation and characterization of particles and polymers, the CAFFFE allows the introduction of higher amounts of sample into the channel in a single run so that semipreparative to preparative separation becomes possible. This was demonstrated by the separation of polymer latex standards.     

A chip system for size separation of macromolecules and particles by hydrodynamic chromatography

For the first time, a miniaturized hydrodynamic chromatography chip system has been developed and tested on separation of fluorescent nanospheres and macromolecules. The device can be applied to size characterization of synthetic polymers, biopolymers, and particles, as an attractive alternative to the classical separation methods such as size exclusion chromatography or field-flow fractionation. The main advantages are fast analysis, high separation efficiency, negligible solvent consumption, and easy temperature control. The prototype chip contains a rectangular flat separation channel with dimensions of 1 microm deep and 1000 microm wide, integrated with a 300-pL injector on a silicon substrate. The silicon microtechnology provides precisely defined geometry, high rigidity, and compatibility with organic solvents or high temperature. All flows are pressure driven, and a specific injection system is employed to avoid excessive sample loading times, demonstrating an alternative way of lab-on-a-chip design. Separations obtained in 3 min show the high performance of the device and are also the first demonstration of flat channel hydrodynamic chromatography in practice.     

Size determination of diesel soot particles using flow and sedimentation field-flow fractionation

The applicability of field-flow fractionation (FFF) was investigated for determination of size and size distribution of diesel soot particles. A sample preparation procedure was developed for FFF analysis where soot particles are recovered from filters in an ethanol bath sonicator, and then they are dispersed in water containing 0.05% Triton X-100 and 0.02% NaN(3). Mean diameters obtained from sedimentation FFF (SdFFF) and flow FFF (FlFFF) agree well with each other and are in good agreement with diameters obtained from photon correlation spectroscopy (PCS) and scanning electron microscopy. The relative error was less than 11%. Data show diesel soot particles have broad size distributions ranging from 0.05 up to ～0.5 μm with the mean diameters between 0.1 and 0.2 μm. The use of FlFFF is more convenient as FlFFF fractograms can be converted directly to size distributions, while the conversion of the SdFFF fractogram needs the particle density information. The density needed for SdFFF analysis was obtained by combining the SdFFF retention data with the PCS size data. For samples whose density is known, SdFFF may be more useful as SdFFF provides a wider dynamic range than FlFFF under constant field strength.     

High-velocity transport of nanoparticles through 1-D nanochannels at very large particle to channel diameter ratios

We explore the possibility of generating high-velocity flows of nanoparticles through flat-rectangular nanochannels, which are only 50% deeper than the diameter of the particles. Using the shear-driven flow principle, 200-nm particles can, for example, be transported through a 300-nm-deep channel at velocities up to 35 mm/s (upper limit of our current setup). Working under high-pH conditions, the velocity of the carboxylated nanoparticles still respects the small-molecule velocity law, despite the high degree of confinement to which the particles are subjected. The high degree of confinement is also found to lead to a reduced band broadening. When injecting sharply delimited particle plugs, the plate heights observed for the flow of 0.2-microm particles through a 0.3-microm channel (with plate heights of the order of 1-2 microm) are, for example, approximately 1 order of magnitude smaller than for the flow of 1.0-microm particles through a 1.4-microm channel. It is also found that the band broadening is, within its statistical variation, independent of the fluid velocity over a large range of particle velocities (5-35 mm/s). The flow method distinguishes itself from pressure-driven field-flow fractionation and hydrodynamic chromatography in that the mean particle velocity is independent of the particle size over the entire range of possible particle to channel diameter ratios.     

A data analysis algorithm for programmed field-flow fractionation

An algorithm that employs numerical integration for analysis of field-flow fractionation (FFF) data is presented. The algorithm utilizes detector response, field strength, and channel flow rate data, monitored at discrete time intervals during sample elution to generate a distribution of sample components according to particle size or molecular weight. The field strength and channel flow rate may either be held constant or programmed as functions of time, and it is not necessary for these programs to follow specific mathematical functions. If experimental conditions are monitored during a run, the algorithm can account for any deviation from nominal set conditions. The algorithm also allows calculation of fractionating power for the actual conditions as monitored during the run. The method provides greatly increased flexibility in the application of the FFF family of techniques. It removes the limitations on experimental conditions incurred by adherence to analytically available solutions to FFF theory, allowing ad hoc variation of field strength and other experimental parameters as necessary to increase sensitivity and specificity of the method. An implementation of the algorithm is described that is independent of the FFF technique (i.e., independent of field type) and mode of operation. To reduce computation time, it uses mathematical techniques to reduce the required number of numerical integrations. This is of particular importance when the perturbations to ideal FFF theory, such as those due to the effects of hydrodynamic lift forces, particle-wall or particle-particle interactions, and secondary relaxation, necessitate relatively lengthy numerical calculations.     

Correction for particle-wall interactions in the separation of colloids by flow field-flow fractionation

In the characterization of materials by field-flow fractionation (FFF), the experienced analyst understands the importance of incorporating additives in the carrier liquid that minimize or eliminate interactions between the analyte and accumulation wall, particularly in aqueous systems. However, as FFF is applied to more difficult samples, such as those with high surface energies, it is increasingly difficult to find additives that completely eliminate particle-wall interactions. Furthermore, the analyst may wish to use specific conditions that preserve the high surface energy of particles, to study their interaction with other materials through their behavior in the FFF channel. With this in mind, Williams and co-workers developed a model that quantifies the effect of particle-wall interactions in FFF using an empirically determined interaction parameter. In this work, the model is evaluated for the application of flow FFF in carrier liquids of low ionic strength, where particle-wall interactions are magnified. The retention of particles ranging in size from 64 to 1000 nm is measured using a wide range of field strengths and retention levels. The model is found to be generally valid over the entire range, except for minor discrepancies at lower levels of retention. Although retention levels are dramatically affected by particle-wall interactions, the point of steric inversion (500 nm), where the size-based elution order reverses, is not affected. When particle-wall interactions are not accounted for, they lead to a bias in particle sizes calculated from standard retention theory of up to 70%. The model can also be used to refine the measurement of channel thickness, which is important for the accurate conversion of retention parameters to particle sizes. In this work, for example, errors in channel thickness led to systematic errors on the order of 10% in particle diameter.     

Dual-field and flow-programmed lift hyperlayer field-flow fractionation.

Field and flow programming and their combination, dual programming, are shown to extend the particle size range to which a single flow/hyperlayer field-flow fractionation (FFF) run is applicable to approximately 1-50 microns. The rationale for programming flow/hyperlayer FFF (or other forms of lift hyperlayer FFF) is to expand the diameter range of micron size particles that can be resolved in a single run. By contrast, the reason for programming normal-mode FFF, the only kind of programming previously realized in FFF, is to reduce the analysis time of submicron particle samples of considerable size variability. These differences are explained in detail in relationship to the basic mechanisms governing retention in normal, steric, and lift hyperlayer FFF. Experiments are described in which field, flow, and dual programming are used to expand the accessible diameter range of flow/hyperlayer FFF. An example is shown in which 11 sizes of latex microspheres in the 2-48-microns diameter range are separated by dual programming in 11 min.     

Hollow-Fiber flow field-flow fractionation of synthetic polymers in organic solvents

A modified polyacrylonitrile (PAN) hollow-fiber membrane from a commercial source has been applied as the separation channel in flow field-flow fractionation (FFF). With the PAN membrane fiber, the application range of flow FFF could be extended to synthetic polymers that are soluble in a variety of organic solvents. The PAN membrane was shown to be resistant to hydrophobic solvents, such as dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate, and methyl ethyl ketone (MEK), as was illustrated by the successful fractionation of different polymer standards in these solvents. The system performance was assessed using polystyrene (PS) standards with ethyl acetate as the solvent. For a 100 kDa PS standard, the average recoverywas 57%, but for standards with a molar mass of 400 kDa and higher, 100% recovery was obtained. A linear relationship between peak area and injected mass was found. The run-to-run and fiber-to-fiber repeatability was determined using 100- and 400 kDa PS standards. The repeatability appeared to be satisfactory, with relative standard deviations < 2% for the retention times and < 5% for the recoveries of the standards. Plate numbers for the 400 kDa standard on different fibers were in the order of 110. From measurements on the fractionation of ferritin aggregates, it is concluded that the instrumental band-broadening is negligible. For an accurate determination of diffusion coefficients and molecular sizes based on retention times, calibration of the channel with standards appeared to be necessary. However, it was shown that the FFF system could be coupled to a multiangle light scattering (MALS) detector, thus providing an alternative on-line method for calibration. Expressions for the maximum obtainable plate number per unit of time have been derived for a hollow-fiber flow FFF system. It is shown that an increase in the system performance can be expected from a scaling down of the fiber diameter.     

Retention in field-flow fractionation with a moderate nonuniformity in the field force

In some field-flow fractionation (FFF) techniques, the basic analyte-field interaction parameter, λ, is not constant but varies within the channel cross section as a result of the nonuniformity of the force exerted by the field on the analyte. This is the case, for instance, in thermal FFF, because of the temperature dependence of the relevant physicochemical transport parameters. To account for this effect, a new FFF retention model is developed, allowing a linear variation of λ from the accumulation to the depletion wall, which is assumed to describe correctly moderate nonuniformity in λ in the vicinity of the accumulation wall. A methodology for sample characterization on the basis of this model is proposed. It associates λ(app), the apparent λ value derived from the retention ratio by means of the classical retention model, with a specific distance from the accumulation wall. An empirical relationship between that distance and λ(app) is derived. In the high retention limit, it is found that this specific distance is not equal, as sometimes intuitively believed, to the mean distance of the molecule or particle cloud to the accumulation wall but is approximately equal to twice this mean distance. The validity of the proposed approach is checked.     

电磁流量计异径管道的流场仿真研究

采用Fluent软件对圆形截面渐变为矩形截面的异径管道流场进行三维建模和数值仿真,分析了横截面收缩异径管的速度分布和流线,建立了矩形截面部分的长度、宽度、高度与进出口压力损失和中心截面平均速度之间的关系.研究表明,中间矩形截面部分的宽度和高度对进出口压损和中心截面平均速度影响较大,同时横截面积收缩比例太大会导致流场紊乱和回流现象,从而为合理设计局部横截面积收缩的电磁流量测量管道提供了理论依据.     

CONTINUOUS FRACTIONATION OF GLASS MICROSPHERES BY GRAVITATIONAL SEDIMENTATION IN SPLIT-FLOW THIN (SPLITT) CELLS

Split-flow thin (SPLITT) separation cells, consisting of submillimeter thick rectangular channels having now splitters at both inlet and outlet ends, were operated continuously using the earth's gravitational field as a driving force to prepare narrow fractions from polydisperse micron-size glass bead populations. Equations arc shown that make it possible to achieve a binary fractionation around a specified cutoff particle diameter by the control of inlet and outlet flowrates. Using a single separation cell, each narrow fraction was obtained by a two step fractionation. one dividing the panicle population around the upper desired limit and the other around the lower desired limit of particle diameters. The clean fractionation by SPLITT cell operation was verified by scanning electron microscopy, which also provided the mean particle diameter and the coefficient of variation for each fraction. The consistency of size distribution results was also examined by steric field-flow fractionation.     

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.     

Analysis of self-assembled cationic lipid-DNA gene carrier complexes using flow field-flow fractionation and light scattering

Self-assembled cationic lipid-DNA complexes have shown an ability to facilitate the delivery of heterologous DNA across outer cell membranes and nuclear membranes (transfection) for gene therapy applications. While the size of the complex and the surface charge (which is a function of the lipid-to-DNA mass ratio) are important factors that determine transfection efficiency, lipid-DNA complex preparations are heterogeneous with respect to particle size and net charge. This heterogeneity contributes to the low transfection efficiency and instability of cationic lipid-DNA vectors. Efforts to define structure-activity relations and stable vector populations have been hampered by the lack of analytical techniques that can separate this type of particle and analyze both the physical characteristics and biological activity of the resulting fractions. In this study, we investigated the feasibility of flow field-flow fractionation (flow FFF) to separate cationic lipid-DNA complexes prepared at various lipid-DNA ratios. The compatibility of the lipid-DNA particles with several combinations of FFF carrier liquids and channel membranes was assessed. In addition, changes in elution profiles (or size distributions) were monitored as a function of time using on-line ultraviolet, multiangle light scattering, and refractive index detectors. Multiangle light scattering detected the formation of particle aggregates during storage, which were not observed with the other detectors. In comparison to population-averaged techniques, such as photon correlation spectroscopy, flow FFF allows a detailed examination of subtle changes in the physical properties of nonviral vectors and provides a basis for the definition of structure-activity relations for this novel class of pharmaceutical agents.