Size fractionation of aquatic colloids and particles by cross-flow filtration: analysis by scanning electron and atomic force microscopy

The suitability of the combined application of environmental scanning electron microscopy (ESEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) for the evaluation of the ability of cross-flow filtration (CFF) to perform adequate size fractionation of freshwater colloids and particles was examined. ESEM and SEM imaging provided reference images of the CFF-generated fractions and, in estimating the experimental cut-off diameter of the membrane, provided evidence that separation was not consistent with nominal pore sizes of the membranes. However, analysis of the images showed that size distribution of CFF-generated fractions and the estimated cut-off diameter of the membranes were dependent on the advantages and limitations of the two imaging techniques. With both ESEM and SEM, best estimates of size cut-offs were lower than the nominal pore size of the membrane in the case of 0.45 μm membranes, but roughly accurate in the case of 0.1 μm pore size membranes. The results also suggested that the effectiveness of CFF may benefit from a pre-separation step using a minimally perturbing technique such as split thin-flow fractionation. AFM demonstrated the presence of colloids smaller than 50 nm in all fractions including the retentates, showing that CFF fractionation is not fully quantitative and not based on size alone. The results indicate that previous studies investigating trace element partitioning using CFF may need re-evaluation as the importance of particles and large colloids may be over-estimated.     

Novel fabrication of on-column capillary inlet frits through flame induced sintering of stainless steel particles

A novel fritting technology was introduced for the fused-silica capillary. The technique involved sintering of stainless steel (SS) particles at the tip of capillary through flame heating. A simple butane gas based welding torch was used for sintering the SS particles. The new fritting technique, flame induced sintering of SS particles (FIS/SSP), was applied for making frits with different inlet diameters (75 μm, 100 μm, 250 μm and 530 μm). The changes in morphologies of SS particles during sintering process were identified by scanning electron microscopy (SEM). Frits with the length of 0.5-1 mm and capillaries with inner diameter about 50-100 μm were fabricated through suitable selection of experimental conditions (size of SS particles and heating mode). The frits prepared by FIS/SSP technique exhibited adequate separation properties and mechanical strength. Columns packed with C₁₈ particles were stable with these frits in a few important chromatographic operations. Frits prepared by FIS/SSP technique was used in three typical separation modes namely, capillary electrochromatography (CEC), p-assisted CEC (p-CEC) and low pressure liquid chromatography (LPLC). Importantly, no bubble formation was noticed with the frit over a period of one week. A good peak symmetry and high efficiency for separation were obtained using pressure-assisted CEC, p-CEC and low pressure-driven separation modes.     

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.     

Hollow-fiber flow/hyperlayer field-flow fractionation for the size characterization of airborne particle fractions obtained by SPLITT fractionation

Hollow-fiber flow field-flow fractionation (HF FlFFF) was applied for the separation and size characterization of airborne particles which were collected in a municipal area and prefractionated into four different-diameter intervals >5.0, 2.5-5.0, 1.5-2.5, <1.5 microm) by continuous split-flow thin (SPLIIT) fractionation. Experiments demonstrated the possibility of utilizing a hollow-fiber module for the high-performance separation of supramicron-sized airborne particles at steric/hyperlayer operating mode of HF FlFFF. Eluting particles during HF FlFFF separation were collected at short time intervals (approximately 10 s) for the microscopic examination. It showed that particle size and size distributions of all SPLITT fractions of airborne particles can be readily obtained using a calibration and that HF FlFFF can be utilized for the size confirmation of the sorted particle fraction during SPLITT fractionation.     

Assessment of cross-flow filtration for the size fractionation of freshwater colloids and particles

This research has evaluated the ability of cross-flow filtration (CFF) to perform correct size fractionation of natural aquatic colloids (materials from 1 nm to 1 μm in size) and particles (>1 μm) using scanning electron microscopy (SEM) combined with atomic force microscopy (AFM). SEM provided very clear images at high lateral resolution (ca. 2-5 nm), whereas AFM offered extremely low resolution limits (sub-nanometer) and was consequently most useful for studying very small material. Both SEM and AFM were consistent in demonstrating the presence of colloids smaller than 50 nm in all fractions including the retentates (i.e. the fractions retained by the CFF membrane), showing that CFF fractionation is not fully quantitative and not based on size alone. This finding suggests that previous studies that investigated trace element partitioning between dissolved, colloidal and particulate fractions using CFF may need to be re-visited as the importance of particles and large colloids may have been over-estimated. The observation that ultra-fine colloidal material strongly interacted with and completely coated a mica substrate to form a thin film has important potential implications for our understanding of the behaviour of trace elements in aquatic systems. The results suggest that clean, ‘pure’ surfaces are unlikely to exist in the natural environment. As surface binding of trace elements is of great importance, the nature of this sorbed layer may dominate trace element partitioning, rather than the nature of the bulk particle.     

Comparison of particle size and flow rate optimization for chromatography using one-monomer molecularly imprinted polymers versus traditional non-covalent molecularly imprinted polymers

The dependence of enantio-selective chromatographic performance on particle size, as measured by separation factor, was investigated for one-monomer molecularly imprinted polymers (OMNiMIPs) compared to traditionally formed EGDMA/MAA molecularly imprinted polymers (MIPs). Five particle size ranges were compared (<20 μm, 20-25 μm, 25-38 μm, 38-45 μm, and 45-63 μm), revealing that the particle sizes above 25 μm provided the highest separation factor, and thus the best enantiomer separation, for both imprinted polymers. Other chromatographic parameters such as the number of theoretical plates and resolution exhibited only minor changes for the OMNiMIPs as the particle size changed, except for particles 20 μm and below. However, the number of theoretical plates and resolution for EGDMA/MAA are higher for particles in the 20-25 μm range. Thus, chromatographic factors for the EGDMA/MAA polymers are better in this range, despite better enantioselectivity for particle sizes above 25 μm. In contrast, OMNiMIPs generally show the most favorable performance for particle sizes in the 38-45 μm range. It was also found that decreasing flow rate resulted in improved enantioselectivity for both MIPs for all particle sizes.     

Metal associations to microparticles, nanocolloids and macromolecules in compost leachates: Size characterization by asymmetrical flow field-flow fractionation coupled to ICP-MS

A methodological approach based on the size characterization of environmental microparticles (size larger than 1 μm), nanocolloids (1 μm to 15 nm) and macromolecules (lower than 1000 kDa) by asymmetrical flow field-flow fractionation (AsFlFFF), taking advantage of both normal and steric elution modes, is presented. The procedure was optimized to minimize the potential alteration of the size distribution and metal associations of the species characterized. Prior to separation by AsFlFFF, samples are subjected to gravitational settling of the solid suspension, followed by a centrifugation of the settled sample. The comparison between the fractograms of the settled and the centrifuged samples allows the characterization of the microparticles, which are eluted in steric mode in the AsFlFFF system. The characterization of nanocolloids and macromolecules is carried out on the centrifuged sample by applying different operational conditions under normal mode in the AsFlFFF system. A comparison with the conventional frontal filtration through 0.45 μm pore size membranes have shown that filtration removes particles below their nominal pore size, modifying the size distribution of the samples respect to the centrifugation. The methodology proposed has been applied to the size characterization of compost leachates. The contribution of these three differentiated fractions to the mobilization of metals has been determined by coupling the AsFlFFF system to an inductively coupled plasma mass spectrometer (ICP-MS).     

Temporal variability of colloidal material in agricultural storm runoff from managed grassland using flow field-flow fractionation

This paper reports the use of flow field-flow fractionation (FlFFF) to determine the temporal variability of colloidal (<1 μm) particle size distributions in agricultural runoff waters in a small managed catchment in SW England during storm events. Three storm events of varying intensity were captured and the colloidal material in the runoff analysed by FlFFF. The technique had sufficient sensitivity to determine directly the changing colloidal profile over the 0.08–1.0 μm size range in the runoff waters during these storm events. Rainfall, total phosphorus and suspended solids in the bulk runoff samples were also determined throughout one storm and showed significant correlation (P < 0.01) with the amount of colloidal material. Whilst there are some uncertainties in the resolution and absolute calibration of the FlFFF profiles, the technique has considerable potential for the quantification of colloidal material in storm runoff waters.     

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.     

Comparative study of new shell-type,sub-2 μm fully porous and monolith stationary phases,focusing on mass-transfer resistance

Today sub-2 μm packed columns are very popular to conduct fast chromatographic separations. The mass-transfer resistance depends on the particle size but some practical limits exist not to reach the theoretically expected plate height and mass-transfer resistance. Another approach applies particles with shortened diffusion path to enhance the efficiency of separations. In this study a systematical evaluation of the possibilities of the separations obtained with 5 cm long narrow bore columns packed with new 2.6 μm shell particles (1.9 μm nonporous core surrounded by a 0.35 μm porous shell, Kinetex™, Core-Shell), packed with other shell-type particles (Ascentis Express™, Fused-Core), totally porous sub-2 μm particles and a 5 cm long narrow bore monolith column is presented. The different commercially available columns were compared by using van Deemter, Knox and kinetic plots. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Data are presented on polar neutral real-life analytes. Comparison of a low molecular weight compounds (MW = 270–430) and a high molecular weight one (MW ∼ 900) was conducted. This study proves that the Kinetex column packed with 2.6 μm shell particles is worthy of rivaling to sub-2 μm columns and other commercially available shell-type packings (Ascentis Express or Halo), both for small and large molecule separation. The Kinetex column offers a very flat C term. Utilizing this feature, high flow rates can be applied to accomplish very fast separations without significant loss in efficiency.     

Fast gradient screening of pharmaceuticals with 5 cm long, narrow bore reversed-phase columns packed with sub-3 μm core-shell and sub-2 μm totally porous particles

The performance of 5 cm long narrow-bore columns packed with 2.6-2.7 μm core-shell particles and a column packed with 1.7 μm totally porous particles was compared in very fast gradient separations of polar neutral active pharmaceutical compounds. Peak capacities as a function of flow-rate and gradient time were measured. Peak capacities around 160-170 could be achieved within 25 min with these 5 cm long columns. The highest peak capacity was obtained with the Kinetex column however it was found that as the flow-rate increases, the peak capacity of the new Poroshell-120 column is getting closer to that obtained with the Kinetex column. Considering the column permeability, peak capacity per unit time and per unit pressure was also calculated. In this comparison the advantage of sub-3 μm core-shell particles is more significant compared to sub-2 μm totally porous particles. Moreover it was found that the very similar sized (d_p = 2.7 μm) and structured (ρ = 0.63) new Poroshell-120 and the earlier introduced Ascentis Express particles showed different efficiency. Results obtained showed that the 5 cm long narrow bore columns packed with sub-3 μm core-shell particles offer the chance of very fast and efficient gradient separations, thus these columns can be applied for fast screening measurements of routine pharmaceutical analysis such as cleaning validation.     

Creation of regenerated cellulose microspheres with diameter ranging from micron to millimeter for chromatography applications

Regenerated cellulose microspheres (RCM) with different diameters were prepared from cellulose solution using 7 wt% NaOH/12 wt% urea aqueous solvent pre-cooled to −12 °C by the sol–gel transition method via a “green” process. By varying the hydrophile–lipophile balance, the amount of the surfactants, the proportion of the water to the oil phase and the stirring speed, the mean diameter of the cellulose microsphere with nano-scale pore size could be controlled easily from 5 μm to 1 mm. The structure and physicochemical properties of the microspheres were characterized by FT-IR spectroscopy, scanning electron microscopy, X-ray diffraction, mercury intrusion-porosimetry and particle size analyzer. The RCM microspheres exhibited spherical shape with the cellulose II structure. A preparative size-exclusion chromatography (SEC) column packed with the cellulose microspheres was used for the fractionation of a polyethylene oxide (PEO) in water, which indicated high efficiency for the fractionations and a large daily throughput of 4 g. Moreover, they had good adsorption capacity to dye particles through physical interaction. The cellulose microspheres would have potential applications in the fields of purification, separation and fractionation of polymers as chromatography packing and adsorbent both at laboratory and industrial scale.     

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.     

Methods for continuous flow fractionation of microparticles: Outlooks and fields of application

The development of new methods for fractionating particles of a different nature is becoming more important in solving some scientific and technological problems. This paper presents a brief review in the theory and practice of the most common techniques for microparticle fractionation (0.1–100 μm). These are dry and wet sieving, elutriation, sequential filtration, split-flow thin fractionation (SPLITT system), field-flow fractionation (FFF), membrane filtration, and capillary electrophoresis. Special attention is paid to the FFF technique, which offers a unique potential for the separation of different materials, from biopolymers and microorganisms to colloidal and solid particles, and the estimation of their physical properties. An alternative version of sedimentation FFF is described, namely, the fractionation of microparticles in rotating coiled columns. The main advantages and limitations of the methods are revealed and their outlooks and fields of applications are envisaged.     

Hydrodynamic chromatography for the size classification of micron and sub-micron sized packing materials

Hydrodynamic chromatography (HDC) was used as a size classification and purification method for porous bridged ethyl hybrid (BEH) packing materials (particles) in the micron to sub-micron range. Using packed column HDC, a batch of particles with size 0.76 ± 0.26 μm was fractionated to yield classified material of 1.05 ± 0.16 μm, reducing the relative standard deviation from 33% to 15%. Subsequent chromatographic evaluation of this packing material showed significant improvement in column performance and decrease in flow resistance over the unclassified material. Comparing a column packed with the classified versus non-classified material, the effective flow resistance of the two columns was decreased by 58% and the minimum HETP for the packing material was improved from 4 to 2.5 μm.     

Advantages of application of UPLC in pharmaceutical analysis

Ultra Performance Liquid Chromatography (UPLC) is a relatively new technique giving new possibilities in liquid chromatography, especially concerning decrease of time and solvent consumption. UPLC chromatographic system is designed in a special way to withstand high system back-pressures. Special analytical columns UPLC Acquity UPLC BEH C₁₈ packed with 1.7 μm particles are used in connection with this system.The quality control analyses of four pharmaceutical formulations were transferred from HPLC to UPLC system. The results are compared for Triamcinolon cream containing trimacinolone acetonide, methylparaben, propylparaben and triamcinolone as degradation product, for Hydrocortison cream (hydrocortisone acetate, methylparaben, propylparaben and hydrocortisone degradation product), for Indomethacin gel (indomethacin and its degradation products 4-chlorobenzoic acid and 5-methoxy-2-methylindoleacetic acid) and for Estrogel gel (estradiol, methylparaben, propylparaben and estrone as degradation product).The UPLC system allows shortening analysis time up to nine times comparing to the conventional system using 5 μm particle packed analytical columns. In comparison with 3 μm particle packed analytical columns analysis should be shortened about three times. The negative effect of particle decrease is back-pressure increase about nine times (versus 5 μm) or three times (versus 3 μm), respectively. The separation on UPLC is performed under very high pressures (up to 100 MPa is possible in UPLC system), but it has no negative influence on analytical column or other components of chromatographic system. Separation efficiency remains maintained or is even improved. Differences and SST parameters, advantages and disadvantages of UPLC are discussed.     

Critical comparison of performances of superficially porous particles and sub-2 μm particles under optimized ultra-high pressure conditions

The performance of 2.7 μm superficially porous particles at 600 bar and sub-2 μm fully porous particles at 1000 bar were compared by the Poppe plot method. Theoretical Poppe plots were first constructed for each stationary phase to compare their kinetic performance at different analysis times. The theory was then verified by experiments under the optimized conditions identified from the Poppe plot calculation. We found that the 2.7 μm superficially porous particles at 600 bar can provide similar performance compared to the sub-2 μm fully porous particles at ultra-high pressure (1000 bar) when analysis times are very short (e.g. sub-minute). As analysis time increases, the superficially porous particles start to outperform the sub-2 μm particles and can give much higher efficiencies (e.g. > 2 times higher plate count) at very long analysis times (>3 h). The comparison was extended to gradient elution of a mixture of pharmaceutical interest by constructing gradient peak capacity Poppe plots and similar behavior was observed.     

Kinetics of thermal degradation and estimation of lifetime for polypropylene particles: Effects of particle size

The thermal stability and degradation behavior of polypropylene (PP) particles having diameter varying from few micrometers to nanometers were studied by thermogravimetric analysis (TGA). The PP particles of average diameter ∼20 μm, ∼10 μm, ∼5 μm, ∼1 μm and <500 nm were studied over a range of temperature from 25 to 600 °C in N₂ atmosphere and heating rates of 5, 10 and 15 °C/min. Thermal stability of PP particles initially decreases and then increases as particle size further decreases to nanometer scale. The five single heating rate techniques such as Friedman, Freeman-Carroll, Chang, Coats-Redfern and second Kissinger; and three multiple heating rate techniques such as the first Kissinger, Kim-Park and Flynn-Wall were used to compute the kinetic parameters of degradation reaction, e.g., activation energy (E_a), order of reaction (n) and frequency factor [ln(Z)]. The lifetime of macro-, micro- and nanosized PP particles was also estimated by a method proposed by Toop. It was found that the activation energy and lifetime of nanosized PP particles are moderately high compared to the microsized PP particles. Moreover, the decomposition temperature, order of reaction (n), frequency factor [ln(Z)] not only depend on the heating rate and calculation technique but also on the particle size of polymer. The results are compared with macrosized PP.     

HPLC analysis of organic acids using a novel stationary phase

In the present work, a high performance liquid chromatographic method with UV detection for the separation of six organic acids including, tartaric, malic, acetic, lactic, citric and succinic is described.The separation was performed on a novel stationary phase TEKNOKROMA, Tr-010065 Mediterranea sea₁₈ (15 cm × 0.4 cm, i.d. 3 μm) and using water with a 0.1% (v/v) of formic acid as mobile phase. The advantages of this packing over a conventional octadecylsilane (ODS2) column are reported.The method was validated with respect to linearity, limits of detection and repeatabilities within day and between days and satisfactory results were obtained.The proposed method was applied for the determination of these compounds in commercially available white wines. The samples were injected directly without previous treatment. LC-MS was used as a confirmatory technique.     

Practical aspects of fast HPLC separations for pharmaceutical process development using monolithic columns

A large number of samples can be generated during pharmaceutical process development. Fast separation for these samples is usually challenging due to the complexity of sample matrix, which requires high efficiency as well as high speed. Monolithic columns (E. Merck, Germany) were investigated as a possible tool for reducing separation time in reversed-phase HPLC without significantly sacrificing efficiency or resolution. Both van Deemter plots and separations of alkyl benzenes and in-process samples showed that monolithic columns were suitable for fast separations without significantly compromising resolution. Practical parameters including the pressure drop, retention factor, selectivity, and tailing factor of monolithic columns (Chromolith type) were compared to those of conventional YMC 150 mm × 4.6 mm (3-μm particles) and 250 mm × 4.6 mm (5-μm particles) packed columns. The batch-to-batch reproducibility of the 100 mm × 4.6 mm Chromolith columns from five randomly ordered batches was also compared to the 250 mm × 4.6 mm YMC particle-packed columns. Fast and efficient separations of complicated process samples including crude drug substances, reaction mixtures, and crystallized mother liquors were demonstrated for both monolithic columns and conventional packed columns. The analysis times were decreased by three to seven times on the coupled monolithic columns, while maintaining the comparable resolution to typical 5-μm particle-packed 250 mm × 4.6 mm columns.