Gravitational Field-flow Fractionation Research Articles

Human umbilical cord blood cells suffer major modification by fixatives and anticoagulants.

Introduction: Developing techniques for the tagless isolation of homogeneous cell populations in physiological-like conditions is of great interest in medical research. A particular case is Gravitational Field-Flow Fractionation (GrFFF), which can be run avoiding cell fixation, and that was already used to separate viable cells. Cell dimensions have a key role in this process. However, their dimensions under physiological-like conditions are not easily known since the most diffused measurement techniques are performed on fixed cells, and the fixation used to preserve tissues can alter the cell size. This work aims to obtain and compare cell size data under physiological-like conditions and in the presence of a fixative. Methods: We developed a new protocol that allows the analysis of blood cells in different conditions. Then, we applied it to obtain a dataset of human cord blood cell dimensions from 32 subjects, comparing two tubes with anticoagulants (EDTA and Citrate) and two tubes with different preservatives (CellRescue and CellSave). We analyzed a total of 2071 cells by using confocal microscopy via bio-imaging to assess dimensions (cellular and nuclear) and morphology. Results: Cell diameter measured does not differ when using the different anticoagulants, except for the increase reported for monocyte in the presence of citrate. Instead, cell dimensions differ when comparing anticoagulants and cell preservative tubes, with a few exceptions. Cells characterized by high cytoplasm content show a reduction in their size, while morphology appears always preserved. In a subgroup of cells, 3D reconstruction was performed. Cell and nucleus volumes were estimated using different methods (specific 3D tool or reconstruction from 2D projection). Discussion: We found that some cell types benefit from a complete 3D analysis because they contain non-spherical structures (mainly for cells characterized by poly-lobated nucleus). Overall, we showed the effect of the preservatives mixture on cell dimensions. Such an effect must be considered when dealing with problems highly dependent on cell size, such as GrFFF. Additionally, such information is crucial in computational models increasingly being employed to simulate biological events.

Experimental and simulation studies of metal sulfide precipitates separation in copper smelting waste acid using a gravitation field-flow fractionation method

Sulfide precipitation method has been widely used in the removal of arsenic and heavy metals from copper smelting waste acid. Nevertheless, tiny metal sulfide particles are generated during the process, making it difficult to achieve solid-liquid separation as well as solid-solid separation. This study designed an innovative gravitational field-flow fractionation (GFFF) tank to effectively separate fine sulfide precipitate particles of different heavy metals, relying on the difference in particle sizes. To achieve better tank performance, the effects of process conditions on the size variation of precipitate particles produced in metal sulfide precipitation process (Cu(Ⅱ), Zn(Ⅱ), or As(Ⅲ)) were firstly investigated. The results show that supersaturation level, sulfide to metal molar ratio, and pH have significant effects on the precipitate particle size, while reaction time affects slightly. The optimum conditions were identified to achieve the greatest possible differences in particle sizes of the three metal sulfide precipitates. To further analyze its performance, a 3D model of the GFFF tank was built, with the fluid flow field and precipitate particle movement simulated under the optimum conditions. The simulation results demonstrate that the designed GFFF tank can realize efficient separation of fine metal sulfide precipitates for the recovery of valuable metals.

The granule characteristics of yam, sweet potato and tapioca starches determined by gravitational field-flow fractionation

The granule characteristics of yam, sweet potato and tapioca starches determined by gravitational field-flow fractionation

Study on the Dependence of Sun Protection Factor on Particle Size Distribution of Mica Using Gravitational Field‐Flow Fractionation

Sunlight consists of beams of light of broad wavelength ranges, including infrared, visible, and ultraviolet (UV) radiation; X‐rays; and γ‐rays. Although the UV content of sunlight is relatively low, the human body, especially the human skin, is sensitive to UV rays. Mica is known to be effective in protecting the human skin from sunlight and reduces fine lines and pores on the skin. Mica is currently used as a coloring agent in various sunscreen products. Still, there is no detailed report on the effect of mica on the sun protection factor (SPF). In this study, effects of the particle size, particle size distribution, and the content of mica on SPF were studied. Gravitational field‐flow fractionation (GrFFF) was employed to analyze the particle size and the particle size distribution of the mica particles. GrFFF provided size‐based separation of the mica particles and was useful for determining the size distributions and average sizes of the mica particles. It was found that the mixing ratio (in wt %) of 3:3:4 of TiO2:octyl methoxycinnamate:mica yielded the highest SPF. Generally, the SPF tends to increase with increasing particle size of the mica.

Fractionation and characterization of starch granules using field-flow fractionation (FFF) and differential scanning calorimetry (DSC)

Starch is one of the main carbohydrates in food; it is formed by two polysaccharides: amylose and amylopectin. The granule size of starch varies with different botanical origins and ranges from less than 1 μm to more than 100 μm. Some physicochemical and functional properties vary with the size of the granule, which makes it of great interest to find an efficient and accurate size-based separation method. In this study, the full-feed depletion mode of split-flow thin cell fractionation (FFD-SF) was employed for a size-based fractionation of two types of starch granules (corn and potato) on a large scale. The fractionation efficiency (FE) of fraction-a for corn and potato granules was 98.4 and 99.4%, respectively. The FFD-SF fractions were analyzed using optical microscopy (OM) and gravitational field-flow fractionation (GrFFF). The respective size distribution results were in close agreement for the corn starch fractions, while they were slightly different for the potato starch fractions. The thermal properties of FFD-SF fractions were analyzed, and the results for the potato starch showed that the peak temperature of gelatinization (Tp) slightly decreases as the size of the granules increases. Additionally, the enthalpy of gelatinization (ΔH) increases when the granule size increases and shows negative correlation with the gelatinization range (ΔT).

Gravitational field flow fractionation: Enhancing the resolution power by using an acoustic force field

An acoustic field flow fractionation (FFF) device was developed to fractionate a micro-particle mixture on the basis of the particle diameter using an acoustic force field in a carrier liquid flow. In the acoustic FFF channel used in the device, ultrasound waves generated from piezoelectric transducers driven by a sinusoidal signal of 2.02 Mhz propagated into the carrier liquid flow and built up a quarter-wavelength ultrasound standing wave field across the channel height. It was experimentally demonstrated that the acoustic field with a pressure node plane at the bottom surface of the channel reduced the thickness of the particle diffusion layer in a stagnant liquid proportional to the applied voltage driving the piezoelectric transducer. In the size-dependent particle separation, the particle mixture flowing through the acoustic FFF channel experienced an acoustic radiation force in the gravitational direction. As a result, suppressing the diffusion of small particles, particles were transported along the bottom surface of the channel with the local velocity of the carrier liquid at the particle center. The developed acoustic FFF device successfully fractionated a fluorescent micro-particle mixture (1, 3, 5, and 10 μm diameter), whereas the 3 and 5 μm particles were not fractionated in the FFF device using only the gravitational force field due to the diffusion of 3 μm particles.

Optimization of experimental conditions for the separation of polystyrene particles by gravitational field-flow fractionation

Optimization of experimental conditions for the separation of polystyrene particles by gravitational field-flow fractionation

A new analytical platform based on field-flow fractionation and olfactory sensor to improve the detection of viable and non-viable bacteria in food.

An integrated sensing system is presented for the first time, wherea metal oxide semiconductor sensor-based electronic olfactory system (MOS array), employed for pathogen bacteria identification based on their volatile organic compound (VOC) characterisation, is assisted by a preliminary separative technique based on gravitational field-flow fractionation (GrFFF). In the integrated system, a preliminary step using GrFFF fractionation of a complex sample provided bacteria-enriched fractions readily available for subsequent MOS array analysis. The MOS array signals were then analysed employing a chemometric approach using principal components analysis (PCA) for a first-data exploration, followed by linear discriminant analysis (LDA) as a classification tool, using the PCA scores as input variables. The ability of the GrFFF-MOS system to distinguish between viable and non-viable cells of the same strain was demonstrated for the first time, yielding 100% ability of correct prediction. The integrated system was also applied as a proof of concept for multianalyte purposes, for the detection of two bacterial strains (Escherichia coli O157:H7 and Yersinia enterocolitica) simultaneously present in artificially contaminated milk samples, obtaining a 100% ability of correct prediction. Acquired results show that GrFFF band slicing before MOS array analysis can significantly increase reliability and reproducibility of pathogen bacteria identification based on their VOC production, simplifying the analytical procedure and largely eliminating sample matrix effects. The developed GrFFF-MOS integrated system can be considered a simple straightforward approach for pathogen bacteria identification directly from their food matrix. Graphical abstract An integrated sensing system is presented for pathogen bacteria identification in food, in which field-flow fractionation is exploited to prepare enriched cell fractions prior to their analysis by electronic olfactory system analysis.

Effects of comprehensive function of factors on retention behavior of microparticles in gravitational field-flow fractionation.

Gravitational field-flow fractionation (GrFFF) is a useful technique for separation and characterization for micrometer-sized particles. Elution behavior of micrometer-sized particles in GrFFF was researched in this study. Particles in GrFFF channel are subject to hydrodynamic lift forces (HLF), fluid inertial forces and gravity, which drive them to different velocities by carrier flow, resulting in a size-based separation. Effects of ionic strength, flow rate and viscosity as well as methanol were investigated using polystyrene latex beads as model particles. This study is devoted to experimental verification of the effect of every factor and their comprehensive function. All experiments were performed to show isolated influence of every variable factor. The orthogonal design test was used to evaluate various factors comprehensively. Results suggested that retention ratio of particles increases with increasing flow rate or the viscosity of carrier liquid by adjusting external forces acting on particles. In addition, retention ratio increases as ionic strength decreases because of decreased electrostatic repulsion between particles and channel accumulation wall. As far as methanol, there is no general trend due to the change of both density and viscosity. On the basis of orthogonal design test it was found that viscosity of carrier liquid plays a significant role in determining resolution of micrometer-sized particles in GrFFF.

Effects of carrier liquid and flow rate on the separation in gravitational field-flow fractionation

Gravitational field-flow fractionation is the simplest field-flow fractionation technique in terms of principle and operation. The earth' s gravity is its external field. Different sized particles are injected into a thin channel and carried by carrier fluid. The different velocities of the carrier liquid in different places results in a size-based separation. A gravitational field-flow fractionation (GrFFF) instrument was designed and constructed. Two kinds of polystyrene (PS) particles with different sizes (20 µm and 6 µm) were chosen as model particles. In this work, the separation of the sample was achieved by changing the concentration of NaN3, the percentage of mixed surfactant in the carrier liquid and the flow rate of carrier liquid. Six levels were set for each factor. The effects of these three factors on the retention ratio (R) and plate height (H) of the PS particles were investigated. It was found that R increased and H decreased with increasing particle size. On the other hand, the R and H increased with increasing flow rate. The R and H also increased with increasing NaN3 concentration. The reason was that the electrostatic repulsive force between the particles and the glass channel wall increased. The force allowed the samples approach closer to the channel wall. The results showed that the resolution and retention time can be improved by adjusting the experimental conditions. These results can provide important values to the further applications of GrFFF technique.

Retention behavior of microparticles in gravitational field-flow fractionation (GrFFF): Effect of ionic strength

Retention behavior of micron-sized particles in gravitational field-flow fractionation (GrFFF) was studied in this study. Effects of ionic strength and flow rate as well as the viscosity of the GrFFF carrier liquid was investigated on the size-based selectivity (Sd), retention ratio (R), and plate height (H) of micron-sized particles using polystyrene latex beads as model particles. It was found that the retention ratio of microparticles increases with increasing flow rate or the viscosity of the carrier liquid as the particles are forced away from the accumulation wall by increased hydrodynamic lift forces (HLF). On the other hand, the retention time increases (retention ratio decreases) with increasing ionic strength of the carrier liquid at the same flow rate, due to decreased repulsive interaction between the particles and the channel accumulation wall (glass in this study) allowing the particles approach closer to the wall. Results suggest the ionic strength of the carrier liquid plays a critical role in determining retention of microparticles in GrFFF as well as the viscosity or the flow rate of the carrier liquid. It was found that the resolution and the separation time could be improved by increasing the carrier viscosity and by carefully adjusting the ionic strength of the carrier liquid.

Gravitational Field-Flow Fractionation Devices Fabricated via a Hot Embossing/Thermal Bonding Method

A novel hot embossing/low temperature ethanol solvent bonding method for the fabrication of polymethylmethacrylate (PMMA) field flow fractionation devices has been developed. The separation channel on a PMMA substrate was generated by a hot embossing process without vacuum. Special temperature-pressure profiles were used to analyze the influence of the hot embossing parameters. After the hot embossing process, ethanol solvent bonding was used to seal the separation channel on the PMMA substrate. The experimental results show that the bonding strength with ethanol solvent bonding at 35 °C (aspect ratio (depth/width): 0.043) is 3.05 MPa, and the deformation percentage is very low (0.54%). A burst pressure test indicated that the as-prepared PMMA gravitational field flow fractionation device has a very high burst pressure. Furthermore, the higher resolution of the as-prepared PMMA gravitational field flow fractionation device in the separation of wheat and starch particles shows that the hot embossing/low temperature ethanol solvent bonding technique will have potential commercial value.

Monitoring of Mixed Culture of Saccharomyces cerevisiae and Acetobacter aceti Using Gravitation Field-flow Fractionation and Gas Chromatography

Department of Biological Science, Hannam University, Daejeon 305-811, KoreaReceived August 27, 2013, Accepted September 17, 2013Key Words : Gravitational field-flow fractionation (GrFFF), Gas Chromatography (GC), Mixed culture, Sac-charomyces cerevisiae (S. cerevisiae), Acetobacter aceti (A. aceti)Saccaromyces cerevisia (S. cerevisiae) is a species ofyeast, which is widely used in wine-making and bread-baking. Acetobacter aceti (A. aceti) is used for production ofvinegar.

중력 장-흐름 분획법을 이용한 느타리버섯 포자의 분리 및 성장 모니터링

중력 장-흐름 분획법 (GrFFF)은 외부장을 중력으로 사용하며 마이크론 크기의 입자들을 분리하고 그들의 특성을 분석하는 데에 유용한 기술이다. 본 연구에서는 느타리버섯 포자들을 크기에 따라 분리하고, 그들의 성장을 모니터 하기 위하여 GrFFF를 응용하였다. 느타리버섯의 포자는 부드러운 표면과 타원체의 모양을 가지는데, 크기는 장축이 약 <TEX>$5{\sim}12{\mu}m$</TEX>, 단축이 약 <TEX>$3{\sim}4{\mu}m$</TEX>의 범위에 있음을 광학현미경을 통하여 확인하였다. 느타리버섯 포자의 분리를 위한 최적유속을 찾기 위하여 0.5~1 mL/min 범위에서 GrFFF 채널 유속을 변화하였다. 또한 성장과정에 미치는 포자 크기의 영향을 알아보기 위하여 최적조건에서 얻은 GrFFF fractogram으로부터 3개의 fraction을 분획하여, 이들을 동일조건에서 30일간 배양하였다. 그 결과, GrFFF fractogram의 중간 부분에서 수집한 포자들, 즉, 중간크기를 가지는 포자들이 fractogram의 앞이나 뒤 부분에서 수집한 포자들보다 상대적으로 더 빨리 성장함을 확인하였다. Gravitational field-flow fractionation (GrFFF) is a separation technique that utilizes earth's gravity as the external field. GrFFF is a convenient tool for the size and/or density-based separation of micron-sized particles of various origins. In this study, GrFFF was employed for size-based separation of oyster mushroom spores. Oyster mushroom spores have smooth surface and are of cylindrical to narrow kidney-shapes with 5 to 12 im in longer dimension and 3 to 4 im in shorter dimension, as was confirmed by optical microscope (OM). GrFFF conditions were optimized for separation and characterization of spores by varying the channel flow rate from 0.5 to 1 mL/min. During the GrFFF elution of the spores, 3 fractions were collected to confirm the growth of oyster mushroom spore. The collected fractions were incubated for 30 days in water to examine the influence of the size on the growth of the spores. Results suggested that the oyster mushroom spores collected at the middle part of the GrFFF fractogram grew faster than those collected at the beginning or at the end of the fractogram.

Field-Flow Fractionation and Hydrodynamic Chromatography on a Microfluidic Chip

We present gravitational field-flow fractionation and hydrodynamic chromatography of colloids eluting through 18 μm microchannels. Using video microscopy and mesoscopic simulations, we investigate the average retention ratio of colloids with both a large specific weight and neutral buoyancy. We consider the entire range of colloid sizes, including particles that barely fit in the microchannel and nanoscopic particles. Ideal theory predicts four operational modes, from hydrodynamic chromatography to Faxén-mode field-flow fractionation. We experimentally demonstrate, for the first time, the existence of the Faxén-mode field-flow fractionation and the transition from hydrodynamic chromatography to normal-mode field-flow fractionation. Furthermore, video microscopy and simulations show that the retention ratios are largely reduced above the steric-inversion point, causing the variation of the retention ratio in the steric- and Faxén-mode regimes to be suppressed due to increased drag. We demonstrate that theory can accurately predict retention ratios if hydrodynamic interactions with the microchannel walls (wall drag) are added to the ideal theory. Rather than limiting the applicability, these effects allow the microfluidic channel size to be tuned to ensure high selectivity. Our findings indicate that particle velocimetry methods must account for the wall-induced lag when determining flow rates in highly confining systems.

New Approaches to the Kinetic Study of Alcoholic Fermentation by Chromatographic Techniques

The kinetics of the fermentation process has gained increasing interest, not only in the scientific community, but in the industrial world as well. Information concerning the improvement of batch fermentation performance may potentially be valuable for the designing of scale-up processes. Intensive studies have been conducted with the use of various chromatographic techniques, such as conventional gas chromatography, reversed-flow gas chromatography (RFGC), high-performance liquid chromatography, field-flow fractionation and others. In the present study, specific focus is placed on the employment of RFGC, a method that can successfully be applied for the determination of physicochemical quantities, such as reaction rate constants and activation energies, at each phase of the alcoholic fermentation. In contrast to conventional chromatographic techniques, RFGC can lead to substantial information referring to the evaluation of fermentation kinetics at any time of the process. Moreover, gravitational field-flow fractionation, a sub-technique of field-flow fractionation, presents the ability to monitor the proliferation of Saccharomyces cerevisiae cells through their elution profiles that can be related to the different cell growth stages. The combination of the two techniques can provide important information for kinetic study and the distinction of the growth phases of yeast cell proliferation during alcoholic fermentations conducted under different environmental conditions.

Gravitational field-flow fractionation integrated with chemiluminescence detection for a self-standing point-of-care compact device in bioanalysis

A "Point-Of-Care-Testing" (POCT) system relies on portable and simply operated self-standing analytical devices. To fulfill diagnostic requirements, the POCT system should provide highly sensitive simultaneous detection of several biomarkers of the pathology of interest (multiplexing) in a short assay time. One of the main unsolved issues in POCT device development is the integration of pre-analytical sample preparation procedures in the miniaturized device. In this work, an integrated POCT system based on gravitational field-flow fractionation (GrFFF) and chemiluminescence (CL) detection is presented for the on-line sample pre-analytical treatment and/or clean-up and analysis of biological fluids. As a proof of principle for the new GrFFF-CL POCT system, the automatic on-line analysis of plasma alkaline phosphatase activity, a biomarker of obstructive liver diseases and bone disorders, starting from whole blood samples was developed. The GrFFF-CL POCT system was able to give quantitative results on blood samples from control and patients with low sample volume (0.5 μL) and reagent consumption, short analysis time (10 minutes), high reproducibility and with a linear range of 50-1400 IU L(-1). The system can be easily applied to on-line prepare plasma from whole blood for other clinical biomarkers and for other assay formats, based on immunoassay or DNA hybridization.

A tag-less method for direct isolation of human umbilical vein endothelial cells by gravitational field-flow fractionation

The analysis of cellular and molecular profiles represents a powerful tool in many biomedical applications to identify the mechanisms underlying the pathological changes. The improvement of cellular starting material and the maintenance of the physiological status in the sample preparation are very useful. Human umbilical vein endothelial cells (HUVEC) are a model for prediction of endothelial dysfunction. HUVEC are enzymatically removed from the umbilical vein by collagenase. This method provides obtaining a good sample yield. However, the obtained cells are often contaminated with blood cells and fibroblasts. Methods based on negative selection by in vitro passages or on the use of defined marker are currently employed to isolate target cells. However, these approaches cannot reproduce physiological status and they require expensive instrumentation. Here we proposed a new method for an easy, tag-less and direct isolation of HUVEC from raw umbilical cord sample based on the gravitational field-flow fractionation (GrFFF). This is a low-cost, fully biocompatible method with low instrumental and training investments for flow-assisted cell fractionation. The method allows obtaining pure cells without cell culture procedures as starting material for further analysis; for example, a proper amount of RNA can be extracted. The approach can be easily integrated into clinical and biomedical procedures.

Application of Gravitational Field-Flow Fractionation (GrFFF) for Monitoring of Clustering Behavior of Staphylococcus aureus

Department of Biotechnology, Hannam University, Daejeon 305-811, KoreaReceived October 21, 2011, Accepted December 13, 2011Key Words : Gravitational field-flow fractionation (GrFFF), Staphylococcus aureus, Clustering behaviorStaphylococcus aureus (S. aureus) is a resident micro-organism, which causes toxic shock syndrome by foodpoisoning and some nosocomial diseases through intra-vascular catheter. The shape of S. aureus is spherical withdiameter of around 1 µm. They tend to clump together toform aggregates and eventually large grape-like clusters.

중력 장-흐름 분획법을 이용한 전분 입자의 swelling에 관한 연구

물의 흡수에 의한 전분알갱이의 swelling은 물과 친화력이 있는 새로운 부분의 노출에 의하여 구조적인 변화로써 점진적으로, 때로는 갑작스럽게 변화를 일으킨다. 이러한 이유로 인해서, 물과의 접촉 시간에 따르는 전분 알갱이의 크기 또는 모양의 변화에 관심을 가진다. 중력 장-흐름 분획법(gravitation field-flow fractionation, GrFFF)은 마이크론 범위의 크기를 가지는 입자들을 분리하는 데에 유용한 분리기술이다. 본 연구에서는 감자와 고구마 전분알갱이들의 크기와 크기 분포도를 결정하는 데에 있어서 GrFFF의 응용가능성을 검사하고자 한다. GrFFF를 이용하여 물과의 접촉 시간에 따르는 전분 알갱이들의 크기분포도 변화를 모니터하였다. GrFFF로부터 얻어진 전분알갱이들의 크기 및 크기분포는 광학현미경(optical microscopy, OM) 결과와 비교하였다. 결과적으로 감자와 고구마 전분의 swelling은 물과의 접촉시간이 증가함에 따라 전분알갱이의 크기가 증가하며, 전분의 종류가 달라지면 swelling 속도가 달라짐을 확인하였다. Swelling of starch granules by water-sorption causes a progressive or sometimes abrupt change in sorption behavior as a result of structural alterations and the possible exposure of new sites with high affinity for water. It is thus of interest to examine the time-dependent change in the size or shape of the starch granules. Gravitational field-flow fractionation (GrFFF) utilizes the earth's gravity as the external field, and is useful for separation of micron-sized particles with larger particles eluting earlier than smaller ones. In this study, GrFFF was used to monitor the swelling of two starch granules, potato starch and sweet potato starch during contact time of 11-12 days at room temperature in water. Results from GrFFF were compared with those obtained from optical microscope (OM). For both starch granules, the mean sizes were increased with time spent in water.