Characterization of polymer nanoparticles by asymmetrical flow field flow fractionation (AF-FFF)

We present the characterization of different polymeric nanoparticles with asymmetrical flow field-flow fractionation (AF-FFF) in different solvents and additional, independent methods such as static and dynamic light scattering (SLS, DLS) in solution and transmission electron microscopy (TEM) and atomic force microscopy (AFM) for the visualization of the nanoparticles on solid substrates. AF-FFF proves to be a powerful technique to determine average sizes of nanoparticles such as multifunctional polyorganosiloxane nanospheres both, in aqueous dispersion and in organic solvents such as toluene. In addition, dye loaded block copolymer vesicles and cylindrical polyelectrolyte type polymacromonomers are successfully analyzed by AF-FFF and the obtained results are compared to the other techniques used.     

Steric-hyperlayer sedimentation field flow fractionation and flow cytometry analysis applied to the study of Saccharomyces cerevisiae

Sedimentation field flow fractionation separation associated with flow cytometry has been used for the characterization of several commercial Saccharomyces cerevisiae yeasts used for wine production. A new type of channel 80 microm thick and new operating conditions, such as sample introduction when field and flow are established and a channel inlet connected to the accumulation wall, were used. Good repeatability (5% RSD) and reduced analysis time (2-10 min) were obtained. The avoidance of the stop-flow relaxation process in conjunction with the use of a channel of reduced thickness has demonstrated that an effective "steric-hyperlayer" mode driving to a major focusing effect of the species in the channel thickness is involved in the elution of the yeast cells. Flow cytometry analyses were performed, and the forward scattering and side scattering yeast characteristics correlation maps were obtained. Field flow fractionation and flow cytometry information obtained indicated that the fractogram profiles of the yeast cell depended not only on the size, but also on the shape and density.     

Capillary magnetic field flow fractionation and analysis of magnetic nanoparticles

This paper reports the purification and analysis of magnetic nanoparticles using capillary magnetic field flow fractionation, which utilizes an applied magnetic field oriented orthogonal to the capillary flow. To validate this approach as a separation method for nanometer-scale particles, samples of magnetic nanoparticles composed of either gamma-Fe2O3 (maghemite) or CoFe2O4 with average diameters ranging from 4 to 13 nm were prepared and characterized by transmission electron microscopy and SQUID magnetometry. Retention of the samples on the capillary was investigated as a function of solvent flow rate and the nanoparticle size and composition; the elution times of the nanoparticles are strongly dependent on their magnetic moments. We demonstrate the use of this method to separate a mixture of nanoparticles into size-monodisperse fractions. The magnetic moments of the particles are calculated based on analysis of the retention parameters and correlate with values obtained in separate SQUID magnetometry measurements.     

Cancer stem cell sorting from colorectal cancer cell lines by sedimentation field flow fractionation

Recently, cancer stem cells (CSCs) have been identified in many types of cancers, such as colorectal cancer (CRC). CSCs seem to be involved in initiation, growth, and tumor metastasis, as well as in radio- and chemotherapy failures. CSCs appears as new biological targets for cancer therapy, requiring the development of noninvasive cell sorting methods. In this study, we used sedimentation field flow fractionation (SdFFF) to prepare enriched populations of CSCs from eight cell lines corresponding to different CRC grades. On the basis of phenotypic and functional characterizations, "hyperlayer" elution resulted in a fraction overexpressing CSC markers (CD44, CD166, EpCAM) for all cell lines. CSCs were eluted in the last fraction for seven out of eight cell lines, but in the first for HCT116. These results suggest, according to the literature, that two different pools of CSCs exist, quiescent and activated, which can both be sorted by SdFFF. Moreover, according to CSC properties, enriched fractions are able to form colonies.     

Characterization of submicrometer aqueous iron(III) colloids formed in the presence of phosphate by sedimentation field flow fractionation with multiangle laser light scattering detection.

Iron colloids play a major role in the water chemistry of natural watersheds and of engineered drinking water distribution systems. Phosphate is frequently added to distribution systems to control corrosion problems, so iron-phosphate colloids may form through reaction of iron in water pipes. In this study, sedimentation field flow fractionation (SdFFF) is coupled on-line with multiangle laser light scattering (MALLS) detection to characterize these iron colloids formed following the oxygenation of iron(II) in the presence of phosphate. The SdFFF-MALLS data were used to calculate the hydrodynamic diameter, density, and particle size distribution of these submicrometer colloids. The system was first verified with standard polystyrene beads, and the results compared well with certified values. Iron(III) colloids were formed in the presence of phosphate at a variety of pH conditions. The colloids' hydrodynamic diameters, which ranged from 218 +/- 3 (pH 7) to 208 +/- 4 nm (pH 10), did not change significantly within the 95% confidence limit. Colloid density did increase significantly from 1.12 +/- 0.01 (pH 7) to 1.36 +/- 0.02 g/mL (pH 10). Iron(III) colloids formed at pH 10 in the presence of phosphate were compared to iron(III) colloids formed without phosphate and also to iron(III) colloids formed with silicate. The iron(III) colloids formed without phosphate or silicate were 0.46 g/mL more dense than any other colloids and were >6 times more narrowly distributed than the other colloids. The data suggest competitive incorporation of respective anions into the colloid during formation.     

Electric circuit model for electrical field flow fractionation

In electrical field flow fractionation (EFFF or ElFFF), an electric potential is applied across a narrow gap filled with a weak electrolyte fluid. Charge buildup at the two poles (electrodes) and the formation of an electric double layer shields the channel, making the effective field in the bulk fluid very weak. Recent computational research suggests that pulsed field protocols, however, should improve retention and may enhance separation in EFFF through systematic disruptions of the double layer resulting in a stronger effective field in the bulk fluid. Improved retention has already been demonstrated experimentally. Accurate modeling and subsequent device optimization and design, however, depends, in part, on formulating a suitable model for the capacitative response of the channel and double layer at the electrode surfaces. Early models do not correctly describe experimentally observed current-time response and are not physically meaningful even when accurate mathematical fits of the data are realized. A new model and conceptual framework based on electrical resistance and capacitance variations of the double layer is suggested here. Physical interpretations of the electrical response have been developed and compared to published experimental data sets.     

Geometric scaling effects in electrical field flow fractionation. 2. Experimental results

Geometric scaling of microelectrical field flow fractionation (micro-EFFF) systems is investigated experimentally and compared to theory and to macroscale EFFF systems. Experimental results are presented to demonstrate that the miniaturized system operates according to the scaling theory associated with the system. Demonstrated improvements in the channels include increased retention and resolution and decreased peak broadening, electrical time constants, relaxation time, power consumption, and sample size. Additionally, scaling effects related to the compression of separation zones in the miniaturized EFFF systems are discussed.     

Geometric scaling effects in electrical field flow fractionation. 1. Theoretical analysis

This work outlines the fundamental scaling laws associated with electrical field flow fractionation channels. Although general FFF theory indicates few advantages from miniaturization, EFFF theory indicates clear advantages to miniaturization of the EFFF channel. Retention, plate heights, resolution, equilibration times, and time constants are examined. The outlined theory predicts scaling advantages in each of these areas after miniaturization. Potential applications, such as the use of these systems for sample preparation in microscale total analysis systems, and improvements associated with these theoretical predictions are also discussed.     

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.     

Asymmetric flow field-flow fractionation of superferrimagnetic iron oxide multicore nanoparticles

Magnetic nanoparticles are very useful for various medical applications where each application requires particles with specific magnetic properties. In this paper we describe the modification of the magnetic properties of magnetic multicore nanoparticles (MCNPs) by size dependent fractionation. This classification was carried out by means of asymmetric flow field-flow fractionation (AF4). A clear increase of the particle size with increasing elution time was confirmed by multi-angle laser light scattering coupled to the AF4 system, dynamic light scattering and Brownian diameters determined by magnetorelaxometry. In this way 16 fractions of particles with different hydrodynamic diameters, ranging between around 100 and 500?nm, were obtained. A high reproducibility of the method was confirmed by the comparison of the mean diameters of fractions of several fractionation runs under identical conditions. The hysteresis curves were measured by vibrating sample magnetometry. Starting from a coercivity of 1.41?kA?m(-1) for the original MCNPs the coercivity of the particles in the different fractions varied from 0.41 to 3.83?kA?m(-1). In our paper it is shown for the first time that fractions obtained from a broad size distributed MCNP fluid classified by AF4 show a strong correlation between hydrodynamic diameter and magnetic properties. Thus we state that AF4 is a suitable technology for reproducible size dependent classification of magnetic multicore nanoparticles suspended as ferrofluids.     

Programmed field decay thermal field flow fractionation of polymers: a calibration method

The universal calibration procedure typical of thermal field flow fractionation (ThFFF) under constant thermal field operation was extended to thermal field programming (TFP) operation. The method requires knowledge of the following: (a) the programming function, which only depends on the thermal field decay function, (b) the physicochemical properties of the solvent, and (c) the calibration plot under varying channel cold wall temperatures (T(c)). Two field flow fractionation field programming conditions, with either a constant or a variable in time carrier flow velocity, are exploited. The method is based on determination, for each retention time position, of the average lambda retention value typical of TFP ThFFF. This parameter is then used to obtain the calibration plot (i.e., the molecular weight of the species as a function of the retention time position) by using the programming function and the calibration plot under varying T(c) values. The procedure approximation errors are also derived as a function of the programming type and solute-solvent system. To properly test the procedure, the calibration plot for the system constituted by polystyrene (PS) in cis-trans Decalin was determined, under varying conditions T(c) and thermal gradients, by using a set of monodisperse PS standards of different molecular weights (M). The procedure was first validated by simulation under two typical cases of TFP ThFFF operation. The approximation errors were found acceptable (in the worse cases, the accuracy in M prediction was 3%) and are in agreement with the theory. The procedure was then experimentally validated under varying programming decay function conditions. The reproducibility and accuracy of the M determination are both better than 2%.     

Miniaturized flow fractionation device assisted by a pulsed electric field for nanoparticle separation

Electric field flow fractionation (EFFF) is a powerful separation technique based on an electrical field perpendicular to a pressure-driven flow. Previous studies of microelectric field flow fractionation (micro-EFFF) indicate that separation performance was limited due to a weak effective electric field caused by polarization layers on the electrode surfaces. In this work, we report on a micro-EFFF device that uses a pulsed voltage scheme to overcome these limitations. The device was fabricated in indium tin oxide (ITO)-coated glass with ITO as electrodes. The effective electric field for pulsed voltage operation was found to be 50-fold stronger when compared with constant voltage operation. A strong influence of pulsed voltage frequency on nanoparticle retention times was observed. Using pulsed voltage, improved separation of polystyrene particles of different surface charge and particle size is demonstrated. Pulsed voltage also offers more parameters compared to the constant voltage mode, e.g., pulse frequency, duty cycle, and waveform to optimize the retention behavior of analytes.     

Characterization of silver nanoparticle products using asymmetric flow field flow fractionation with a multidetector approach--a comparison to transmission electron microscopy and batch dynamic light scattering

Due to the already prevalent and increasing use of silver-nanoparticle (Ag-NP) products and the raised concerns in particular for the aquatic environment, analytical techniques for the characterization of such products are of need. However, because Ag-NP products are of different compositions and polydispersities, analysis especially of the size distribution is challenging. In this work, an asymmetric flow field flow fractionation (A4F) multidetector system (UV/vis, light scattering, inductively coupled plasma mass spectrometry - ICPMS), in combination with a method to distinguish and quantify the particle and dissolved Ag fractions (ICPMS after ultracentrifugation), for the characterization of Ag-NP products with different degrees of polydispersities is presented. For validation and to outline benefits and limitations, results obtained from batch dynamic light scattering (batch-DLS) and transmission electron microscopy (TEM) were compared. With the developed method a comprehensive understanding in terms of dissolved Ag and Ag-NP concentration as well as an element selective, mass- and number particle size distribution (PSD) was obtained. In relation to batch-DLS, the reliability of the data was improved significantly. In comparison to TEM, faster measurement times and the ability to determine the samples directly in dispersions are clearly advantageous. The proposed setup shows potential for a rapid- and reliable characterization method of virtually any polydisperse metallic NP dispersion, many of them available on the market already.     

Application of flow field flow fractionation-ICPMS for the study of uranium binding in bacterial cell suspensions

Field flow fractionation (FFF) is a size-based separation technique applicable to biomolecules, colloids, and bacteria in solution. When interfaced with ICPMS on-line, elemental data can be collected concurrent with size distribution. We employed hyperlayer flow FFF (Fl FFF) methodology to separate cells of Shewanella oneidensis strain MR-1 from exopolymers present in washed cell suspensions. With a channel flow of 4 mL min-1 and a cross-flow of 0.4 mL min-1 cells eluted with a retention time of 4.7 min corresponding to an approximate equivalent spherical cell diameter of 0.8 microm. Cell suspensions were amended with increasing concentrations of U to establish an adsorption isotherm and with fixed U concentrations at varying pH to establish the pH dependence of sorption. A linear sorption isotherm was determined for U solution concentrations of 0.2-16 microM, maximum U sorption occurred at pH 5. A high molecular weight compound, presumably a cell exudate, was identified by Fl FFF-ICPMS. This cell exudate complexed U, and at elevated pH, the exudate appeared to have a greater affinity for U than cell surfaces. Thus, Fl FFF interfaced with ICPMS detection is a powerful analytical technique for metal sorption studies with bacteria; analysis can be carried out on small sample volumes (25 microL) and additional speciation information can be gained because of the versatile Fl FFF separation range and multielement detection capabilities of ICPMS.     

Mouse embryonic stem cell sorting for the generation of transgenic mice by sedimentation field-flow fractionation

Mouse embryonic stem (ES) cells are an important tool for generation of transgenic mice and genetically modified mice. A rapid and efficient separation of ES cells that respects cell integrity, viability, and their developmental potential while also allowing purified ES fraction collection under sterile conditions might be of great interest to facilitate the generation of chimeric animals. In this study, we demonstrated for the first time the effectiveness of a sedimentation field-flow fractionation (SdFFF) cell sorter to provide, with a characteristic DNA content, a purified ES cell fraction and with a high in vivo developmental potential to prepare transgenic mice by generation of chimeras having a high percentage of chimerism.     

The flow field near a venous needle in hemodialysis: A computational study

The vascular access used in hemodialysis can suffer from numerous complications, which may lead to failure of the access, patient morbidity, and significant costs. The flow field in the region of the venous needle may be a source of damaging hemodynamics and hence adverse effects on the fistula. In this study, the venous needle flow has been considered, using three‐dimensional computational methods. Four scenarios where the venous needle flow could potentially influence dialysis treatment outcome were identified and examined: Variation of the needle placement angle (10°, 20°, 30°), variation of the blood flow rate settings (200, 300, 400 mL/min), variation of the needle depth (top, middle, bottom), and the inclusion of a back eye in the needle design. The presence of the needle has significant effect on the flow field, with different scenarios having varying influence. In general, wall shear stresses were elevated above normal physiological values, and increased presence of areas of low velocity and recirculation—indicating increased likelihood of intimal hyperplasia development—were found. Computational results showed that the presence of the venous needle in a hemodialysis fistula leads to abnormal and potentially damaging flow conditions and that optimization of needle parameters could aid in the reduction of vascular access complications. Results indicate shallow needle angles and lower blood flow rates may minimize vessel damage.     

Size separation of supermicrometer particles in asymmetrical flow field-flow fractionation. Flow conditions for rapid elution

The performance of lift-hyperlayer asymmetrical flow field-flow fractionation using rapid elution conditions was tested through the separation of standard polystyrene latex particles of diameters from 2 to 20 microm. Optimization of flowrates was studied not only in order to obtain efficient and rapid separation, but also to work under conditions of various shape and steepness of the axial flow velocity gradient. Using extreme flow conditions, the five widely spaced particle sizes, 20.5-, 15.0-, 9.7-, 5.0-, and 2.0-microm diameter, could be resolved in 6 min, whereas for the narrower size range of 20.5-5.0 microm, 1 min was enough. The size selectivity in the size range 9.7-2.0 microm was studied as a function of flowrates and particle size and was found to be constant. A particle trapping device made it possible to separate particles of sizes > 10 microm, which has previously proven to be difficult in asymmetrical channels.