Magnetic split-flow thin fractionation of magnetically susceptible particles.

We recently built a magnetic separation system to extend the applications of split-flow thin (SPLITT) fractionation to magnetically susceptible particles. Here, we characterize the magnetic SPLITT system using magnetically susceptible particles and ion-labeled particles. The flow axis of separation channel was orientated parallel and perpendicular to gravitational forces to exclude and include, respectively, gravitational effects on separation. Both operating modes were used to test the theory experimentally, with emphasis on the parallel mode. The magnetic susceptibilities of carrier and ion-labeled particles were varied, and various ion-labeled and unlabeled particles were studied experimentally, resulting in successful separation of labeled particles, yeasts, and cells from unlabeled ones. The minimal difference in magnetic susceptibility (delta(chi)) required for complete particle separation was about 1.75 x 10(-5) [cgs], corresponding to about 10(9) labeling ions per particle in this study. The throughput was around 7.2 x 10(8) particles/h using the present setup. Magnetic SPLITT fractionation shows good potential for use in obtaining particles magnetic susceptibilities from a simple theoretical treatment.     

Investigation of red blood cell fractionation by gravitational field-flow fractionation.

Gravitational field-flow fractionation is used for the separation of particles according to their sizes in the range 1-100 microns: larger particles elute before smaller ones. This phenomenon can be explained as a result of the steric exclusion of the particles from the vicinity of the channel walls, and/or hydrodynamic effects supposedly associated with the inertia of the liquid. The method was used for the investigation of red blood cells. The dependence of the retention ratio on the flow-rate, sample volume, concentration of blood and relaxation time was studied. Analysis of fifteen individual fractions by Coulter counter and reinjection of three other fractions were studied in order to verify fractionation of red blood cells.     

Pinched inlet gravitational split-flow thin fractionation of airborne particles and analysis of size dependent level of PCDD/Fs

Fine particles in air have a direct influence on human health because they carry toxic chemicals that can be deposited in the human lung when inhaled. Thus, particle size distribution and size dependent level of contamination of the airborne particles are important parameters for the study and assessment of environmental pollution. In this study, gravitational split-flow thin (SPLITT) fractionation (or GSF), a semi-preparative scale separation technique for particles, was applied for the continuous size sorting of airborne particles collected in urban area. About 2.0 g of airborne particles was fractionated into four different size intervals (<1.5, 1.5-2.5, 2.5-5.0, and >5.0 microm), and the collected fractions were examined by electron microscopy for particle size distribution and analyzed for the size dependent levels of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs). It was found that more than 60% of particles including dissolved matters in weight were smaller than 5.0 microm and they contained more than 86% of the total PCDD/Fs amount in airborne particles.     

Human lymphocyte sorting by gravitational field-flow fractionation

Interest in biological studies on various cell types for many biomedical applications, from research to patient treatments, is constantly increasing. The ability to discriminate (sort) and/or quantify distinct subpopulations of cells has become increasingly important. For instance, not only detection but also the highest depletion of neoplastic cells from normal cells is an important requisite in the autologous transplantation of lymphocytes for blood cancer treatments. In this work, gravitational field-flow fractionation (GrFFF) is shown to be effective for sorting a heterogeneous mixture of human, living lymphocytes constituted of neoplastic B cells from a Burkitt lymphoma cell line and healthy T and B lymphocytes from blood samples. GrFFF does not require the use of fluorescent immunotags for sorting cells, and the sorted cells can be collected for their further characterization. Flow cytometry was used to assess the viability of the cells collected, and to evaluate the cell fractionation achieved. A low amount of neoplastic B lymphocytes (less than 2%) was found in a specific fraction obtained by GrFFF. The high depletion from neoplastic cells (more than 98%) was confirmed by a clonogenicity test.     

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。     

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.     

Optimization of experimental conditions for the separation of small and large starch granules by gravitational field-flow fractionation

Separation of small and large barley starch granules by gravitational field-flow fractionation was investigated from the point of view of sample pre-treatment, amount of injected sample, and elution conditions. The sample pre-treatment study resulted in the conclusion that it is reasonable to soak the starch granules for at least 24 h prior to separation. The experiments with different amounts of injected sample show that it is possible to increase as well as decrease twofold the sample amount usually used without any change in retention ratios. The implementation of flow-rate gradients for elution of the starch granules reduced total separation time. However, the applied flow-rate gradients did not improve the resolution of peaks A and B compared with the generally used constant flow-rate. Thus, for barley starch granules, the constant flow-rates within the range from 0.8 to 1.0 ml/min seem to provide the best compromise of total separation time, peak resolution and instrumental expense.     

General theory for flow optimisation of split-flow thin fractionation

Recently, magnetic split-flow thin (SPLITT) fractionation has been developed to separate macromolecules, colloids, cells and particles. However, the previous theory, developed for an infinitely long channel, needs to be improved to consider the flow transit regimes at both inlet and outlet. In this paper, we describe a new approach to optimising flow-rates for particle separation which considers the effect of flow transit region. Surprisingly, the critical particle migration velocities derived by the present theory are identical to the previous simplified theory. Therefore, the previous simplified theory may have wider application than might have been expected. As a test of our theory, a numerical simulation based on solving Navier-Stokes equations has also been carried out for a magnetic SPLITT device. The trajectory of a particle with the critical migration velocity is exactly as expected by our theory. Following experimental validation, this work will facilitate the design of new SPLITT fractionation systems with smaller aspect ratio.     

Particle magnetic susceptibility determination using analytical split-flow thin fractionation

Magnetic split-flow thin (SPLITT) fractionation is a newly developed S     

Standardless method for quantitative particle-size distribution studies by gravitational field-flow fractionation. Application to silica particles

Summary Gravitational field-flow fractionation is a separative analytical technique which has already proved suitable for quantitative particle-size distribution analysis. One of the most attractive aspects of the technique is that it can allow for direct conversion of fractograms into size distributions of the samples, although retention exhibits substantial dependence on flow rate, compared to other field-flow fractionation methods. It is shown here that conversion of fractograms into quantitative, size-distribution profiles of micron-sized silica particles is possible through gravitational field-flow fractionation in standardless mode. Standardless means that the conversion of fractograms is performed by single-run analysis because all the parameters necessary for the calculations can be obtained, from sample specifications and previous instrumental calibration, by means of semiempirical models. Work partially presented at FFF’98-7^th International Symposium on Field-Flow Fractionation, Salt Lake City, Utah (USA), February 8–11, 1998     

Combination of gravitational SPLITT fractionation and field-flow fractionation for size-sorting and characterization of sea sediment

A combination of gravitational split-flow thin (SPLITT) fractionation and sedimentation/steric field-flow fractionation (Sd/StFFF) has been used for continuous size-sorting of a sediment sample and for size analysis of the collected fractions. An IAEA (International Atomic Energy Agency) sediment material was separated into four size fractions (with theoretical size ranges <1.0, 1.0–3.0, 3.0–5.0, and >5.0 m in diameter) by means of a three-step gravitational SPLITT fractionation (GSF) for which the same GSF channel was used throughout. The GSF fractions were collected and examined by optical microscopy (OM) and by Sd/St FFF. The mean diameters of the GSF fractions measured by OM were within the size interval predicted by GSF theory, despite the theory assuming that all particles are spherical, which is not true for the sediment particles. The Sd/St FFF results showed that retention shifted toward shorter elution time (or larger size) than expected, probably because of the shape effect. The results from GSF, OM, and Sd/StFFF are discussed in detail.     

Size-based speciation of iron in clay mineral particles by gravitational field-flow fractionation with electrothermal atomic absorption spectrometry

Gravitational field-flow fractionation (FFF) coupled to UV and ETAAS detectors has been tested for micron-size particles in the range of 5–20 μm using three Fe-rich clay samples. The iron content estimated after aqua regia extraction was about 20–40 mg kg⁻¹. The ETAAS analysis was performed both off-line from collected fractions and in an online continuous sampling mode using a specially designed flow through vial placed in the autosampler of the ETAAS. Comparison of the direct injection method with total analysis after aqua regia digestion shows that slurry injection of the dilute samples in the gravitational field-flow fractionation (GrFFF) effluent is quite efficient in these samples. In the majority of cases, more than 90% recovery was obtained for the slurry injection method. Fe mass-based particle size distributions and Fe concentration versus particle diameter plots can be generated using certain assumptions. This provides detailed information on size-based speciation of particulate samples. Generally, the Fe concentrations in the particles decreased slightly with an increase in particle size as is often found for soil and sediment samples.     

Elution mode of Pneumocystis carinii cysts in gravitational field-flow fractionation.

The simplest field-flow fractionation technique, i.e. gravitational, was used in an attempt to purify a Pneumocystis carinii cyst suspension. This parasite is an opportunistic invader in immunocompromised patients, especially those suffering from AIDS. The cyst stage is spherical and 5 microns in diameter. Unexpected retention times, not systematically related to the size and the density of the parasite, were obtained under various experimental conditions. When silicone-coated walls were used, Pneumocystis carinii cysts were eluted in the void volume, whereas when uncoated walls were used with a sodium dodecyl sulphate-enriched carrier phase, retention was observed. These phenomena are probably related to the high degree of hydrophobicity of these micrometre-sized biological particles; this degree can be easily determined. The use of the gravitational field-flow fractionation technique can be of a great interest for the development of new methods for diagnostic purposes. Particle-wall interactions and their modifications due to the carrier phase or to the wall treatment can be employed in the search for new bronchoalveolar lavage solutions.     

Separation of living red blood cells by gravitational field-flow fractionation.

The field-flow fractionation technique, using the earth's gravitational field, has been applied to peripheral blood cell populations. A more or less symmetrical, gaussian-like, elution peak is generally observed for the red cell population. The bimodal cell population obtained after a massive transfusion is shown to result in a shoulder on the red blood cell elution profile. In one case where a similar shouldering peak was obtained from a non-transfused donor, the existence of an immunological double population has been demonstrated. This suggests that field-flow fractionation has some potential for complementary biomedical diagnosis.     

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.     

Continuous two-dimensional field-flow fractionation: a novel technique for continuous separation and collection of macromolecules and particles

Instrumental techniques to analyse macromolecular and particulate materials have undergone rapid development in response to the need for high resolution, precise identification and characterization, and enrichment and collection for further analysis. Continuous two-dimensional field-flow fractionation (2D-FFF), which is described in this article, is a novel technique for separation and collection of macromolecules and particles. 2D-FFF is based on the conventional field-flow fractionation principle but with carrier flow in two-dimensions. This overview discusses the principle of the technique, describes the instrumentation and suggests potential applications and further extensions. An overview of the basic field-flow fractionation principle is presented.     

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.     

Size fractionation of marine sediments by pinched inlet gravitational split-flow thin fractionation and the study of size dependent PCDD/Fs concentrations from different bay areas

Pinched inlet gravitational split-flow thin fractionation (PI-GSF) has been applied to the continuous size fractionation of marine sediments in order to study the difference in sediment size distribution and the concentration of PCDD/Fs contained in different particle sizes. A PI-GSF channel, known to improve the separation efficiency by reducing the sample inlet thickness, was utilized to fractionate sediments collected from three different bay areas (Geoje, Ulsan, and Pohang) in Korea into 5 different sub-populations (<2.0, 2.0-5.0, 5.0-10, 10-20, 20-63 microm in diameter). The sorted sediment fractions from PI-GSF were examined using electron microscopy to obtain size distribution and the results showed a variation in particle size distribution between bay areas. When the collected particle fractions were examined for size dependent levels of PCDD/Fs, the concentrations of total PCDD/Fs were shown to be much greater for samples collected close to heavy industry complexes than sediments from bay areas without major industry.