无固定相分离-电感耦合等离子体质谱法在环境中痕量金属纳米颗粒分析中的应用

环境中金属纳米颗粒的分析检测不仅需要关注其浓度和化学组成,还需要对其形状、粒径和表面电荷等进行表征.此外,环境中金属纳米颗粒的分析需要解决其低赋存浓度以及复杂基质干扰的难题.无固定相分离技术与电感耦合等离子体质谱(ICP-MS)的在线联用,具有较强的颗粒分离能力和较低的元素检出限,能够快速准确地提供金属纳米颗粒的粒径分...     

Computer simulation of particle separation based on non-equilibrium swelling

Steric/hyperlayer field-flow fractionation (FFF) is an established analytical technique for separating and characterizing particles in the 1-100 microns diameter range. The separation can be based on differences in size, density, shape and mechanical properties of the particles. In the course of an analysis of the water transporter system of Chinese hamster ovary (CHO) cells and one of their high permeability mutants, the first successful attempt was made to use the steric/hyperlayer FFF system for the purpose of separating particles based on a time-dependent property, namely, the differential swelling of the two cell types. The present study was undertaken to simulate numerically the separation and suggest selection of operating conditions to minimize repetitive experiments. The computer simulation was developed using Maple V, a symbolic computing environment. It is shown that the model is able to predict an optimal velocity of carrier buffer that maximizes resolution. Predicted velocity/resolution pairs are in good agreement with available experimental data. Empirical models for the lift forces encountered in such FFF experiments, and for the zone broadening observed in work with cell sized particles, form the basis for this model.     

Sonication effect on cellular material in sedimentation and gravitational field flow fractionation

Sonication procedures are generally used prior to field flow fractionation (FFF) separation in order to produce suspensions without aggregates. Yeast cells manufactured in active dry wine yeast (ADWY) were placed in an ultrasound water bath in order to disrupt possible clumps and to obtain a single-cell suspension to be used in optimal conditions during fermentation processes. In order to determine whether this sample preparation procedure meets absolute needs, different yeast samples before and after sonication were analysed by two field flow fractionation techniques. It is shown that 2 min of sonication in the sample preparation process is sufficient to obtain an optimal dispersion of the yeast cells, that is, without critical percentage of aggregates. To demonstrate this effect, photographs of the yeast cell suspensions were performed with non-sonicated and sonicated yeast sample dispersion. The resulting data are compared with the elution profiles obtained from the two different FFF techniques. It is demonstrated that fractogram profiles prove the effectiveness of sonication methodologies.     

Characterization of silver nanoparticles using flow-field flow fractionation interfaced to inductively coupled plasma mass spectrometry

The ability to detect and identify the physiochemical form of contaminants in the environment is important for degradation, fate and transport, and toxicity studies. This is particularly true of nanomaterials that exist as discrete particles rather than dissolved or sorbed contaminant molecules in the environment. Nanoparticles will tend to agglomerate or dissolve, based on solution chemistry, which will drastically affect their environmental properties. The current study investigates the use of field flow fractionation (FFF) interfaced to inductively coupled plasma-mass spectrometry (ICP-MS) as a sensitive and selective method for detection and characterization of silver nanoparticles. Transmission electron microscopy (TEM) is used to verify the morphology and primary particle size and size distribution of precisely engineered silver nanoparticles. Subsequently, the hydrodynamic size measurements by FFF are compared to dynamic light scattering (DLS) to verify the accuracy of the size determination. Additionally, the sensitivity of the ICP-MS detector is demonstrated by fractionation of μg/L concentrations of mixed silver nanoparticle standards. The technique has been applied to nanoparticle suspensions prior to use in toxicity studies, and post-exposure biological tissue analysis. Silver nanoparticles extracted from tissues of the sediment-dwelling, freshwater oligochaete Lumbriculus variegatus increased in size from approximately 31-46nm, indicating a significant change in the nanoparticle characteristics during exposure.     

Osmolarity effects on red blood cell elution in sedimentation field-flow fractionation.

Field-flow fractionation (FFF) is an analytical technique particularly suitable for the separation, isolation, and characterization of macromolecules and micrometer- or submicrometer-sized particles. This chromatographic-like methodology can modulate the retention of micron-sized species according to an elution mode described to date as "steric hyperlayer". In such a model, differences in sample species size, density, or other physical parameters make particle selective elution possible depending on the configuration and the operating conditions of the FFF system. Elution characteristics of micron-sized particles of biological origin, such as cells, can be modified using media and carrier phases of different osmolarities. In these media, a cells average size, density, and shape are modified. Therefore, systematic studies of a single reference cell population, red blood cells (RBCs), are performed with 2 sedimentation FFF systems using either gravity (GrFFF) or a centrifugational field (SdFFF). However, in all cases, normal erythrocyte in isotonic suspension elutes as a single peak when fractionated in these systems. With carrier phases of different osmolarities, FFF elution characteristics of RBCs are modified. Retention modifications are qualitatively consistent with the "steric-hyperlayer" model. Such systematic studies confirm the key role of size, density, and shape in the elution mode of RBCs in sedimentation FFF for living, micronsized biological species. Using polymers as an analogy, the RBC population is described as highly "polydisperse". However, this definition must be reconsidered depending on the parameters under concern, leading to a matricial concept: multipolydispersity. It is observed that multipolydispersity modifications of a given RBC population are qualitatively correlated to the eluted sample band width.     

Advances in field-flow fractionation for the analysis of biomolecules: instrument design and hyphenation

Field-flow fractionation (FFF) separates analytes by use of an axial channel-flow and a cross-field. Its soft separation capability makes it an ideal tool for initial fractionation of complex mixtures, but large elution volumes and high flow rates have limited its applicability without significant user handling. Recent advances in instrumentation and miniaturization have successfully reduced channel size and elution speed, and thus the volume of each fraction, making it possible to conveniently couple FFF with orthogonal separation techniques for improved resolution. More detailed analysis can also be performed on the fractions generated by FFF by use of diverse analytical techniques, including MS, NMR, and even X-ray scattering. These developmental trends have given FFF more power in the analysis of different types of molecule, and will be the direction of choice for further advances in FFF technology.     

Separation and characterization of nanoparticles in complex food and environmental samples by field-flow fractionation

The thorough analysis of natural nanoparticles (NPs) and engineered NPs involves the sequence of detection, identification, quantification and, if possible, detailed characterization. In a complex or heterogeneous sample, each step of this sequence is an individual challenge, and, given suitable sample preparation, field-flow fractionation (FFF) is one of the most promising techniques to achieve relevant characterization.The objective of this review is to present the current status of FFF as an analytical separation technique for the study of NPs in complex food and environmental samples. FFF has been applied for separation of various types of NP (e.g., organic macromolecules, and carbonaceous or inorganic NPs) in different types of media (e.g., natural waters, soil extracts or food samples).FFF can be coupled to different types of detectors that offer additional information and specificity, and the determination of size-dependent properties typically inaccessible to other techniques. The separation conditions need to be carefully adapted to account for specific particle properties, so quantitative analysis of heterogeneous or complex samples is difficult as soon as matrix constituents in the samples require contradictory separation conditions. The potential of FFF analysis should always be evaluated bearing in mind the impact of the necessary sample preparation, the information that can be retrieved from the chosen detection systems and the influence of the chosen separation conditions on all types of NP in the sample. A holistic methodological approach is preferable to a technique-focused one.     

Novel Methods to Manipulate Autolysis in Sparkling Wine: Effects on Yeast

Sparkling wine made by the traditional method (Méthode Traditionelle) develops a distinct and desirable flavour and aroma profile attributed to proteolytic processes during prolonged ageing on lees. Microwave, ultrasound and addition of β-glucanase enzymes were applied to accelerate the disruption of Saccharomyces cerevisiae, and added to the tirage solution for secondary fermentation in traditional sparkling winemaking. Scanning electron microscopy and flow cytometry analyses were used to observe and describe yeast whole-cell anatomy, and cell integrity and structure via propidium iodide (PI) permeability after 6-, 12- and 18-months post-tirage. Treatments applied produced features on lees that were distinct from that of the untreated control yeast. Whilst control yeast displayed budding cells (growth features) with smooth, cavitated and flat external cell appearances; microwave treated yeast cells exhibited modifications like ‘doughnut’ shapes immediately after treatment (time 0). Similar ‘doughnut’-shaped and ‘pitted/porous’ cell features were observed on progressively older lees from the control. Flow cytometry was used to discriminate yeast populations; features consistent with cell disruption were observed in the microwave, ultrasound and enzyme treatments, as evidenced by up to 4-fold increase in PI signal in the microwave treatment. Forward and side scatter signals reflected changes in size and structure of yeast cells, in all treatments applied. When flow cytometry was interpreted alongside the scanning electron microscopy images, bimodal populations of yeast cells with low and high PI intensities were revealed and distinctive ‘doughnut’-shaped cell features observed in association with the microwave treatment only at tirage, that were not observed until 12 months wine ageing in older lees from the control. This work offers both a rapid approach to visualise alterations to yeast cell surfaces and a better understanding of the mechanisms of yeast lysis. Microwave, ultrasound or β-glucanase enzymes are tools that could potentially initiate the release of yeast cell compounds into wine. Further investigation into the impact of such treatments on the flavour and aroma profiles of the wines through sensory evaluation is warranted.     

Picoliter-volume aqueous droplets in oil: electrochemical detection and yeast cell electroporation

An electrochemical detection method was introduced for aqueous droplet analysis in oil phase of microfluidic devices. This method is based on the electrochemical signal difference between aqueous and oil. Applying a low alternating current (AC) voltage to a couple of Au microelectrodes, this method can offer size information and ion concentration range from 0.02 mmol/L to 1 mol/L of tens of picoliter to nanoliter aqueous droplets. Alternatively, applying a relative high AC voltage (18 Vpp) at a frequency of 1 kHz leads to electroporation of yeast cells encapsulated into picoliter droplets. We believe that this simple technique is useful for a number of aqueous droplet-based chemical and biological analyses as well as cell electroporation.     

Coupling gravitational and flow field-flow fractionation, and size-distribution analysis of whole yeast cells

This work continues the project on field-flow fractionation characterisation of whole wine-making yeast cells reported in previous papers. When yeast cells are fractionated by gravitational field-flow fractionation and cell sizing of the collected fractions is achieved by the electrosensing zone technique (Coulter counter), it is shown that yeast cell retention depends on differences between physical indexes of yeast cells other than size. Scanning electron microscopy on collected fractions actually shows co-elution of yeast cells of different size and shape. Otherwise, the observed agreement between the particle size distribution analysis obtained by means of the Coulter counter and by flow field-flow fractionation, which employs a second mobile phase flow as applied field instead of Earths gravity, indicates that yeast cell density can play a major role in the gravitational field-flow fractionation retention mechanism of yeast cells, in which flow field-flow fractionation retention is independent of particle density. Flow field-flow fractionation is then coupled off-line to gravitational field-flow fractionation for more accurate characterisation of the doubly-fractionated cells. Coupling gravitational and flow field-flow fractionation eventually furnishes more information on the multipolydispersity indexes of yeast cells, in particular on their shape and density polydispersity.     

Field-flow fractionation in bioanalysis: A review of recent trends

Field-flow fractionation (FFF) is a mature technique in bioanalysis, and the number of applications to proteins and protein complexes, viruses, derivatized nano- and micronsized beads, sub-cellular units, and whole cell separation is constantly increasing. This can be ascribed to the non-invasivity of FFF when directly applied to biosamples. FFF is carried out in an open-channel structure by a flow stream of a mobile phase of any composition, and it is solely based on the interaction of the analytes with a perpendicularly applied field. For these reasons, fractionation is developed without surface interaction of the analyte with packing or gel media and without using degrading mobile phases. The fractionation device can be also easily sterilized, and analytes can be maintained under a bio-friendly environment. This allows to maintain native conditions of the sample in solution.In this review, FFF principles are briefly described, and some pioneering developments and applications in the bioanalytical field are tabled before detailed report of most recent FFF applications obtained also with the hyphenation of FFF with highly specific, sensitive characterization methods. Special focus is finally given to the emerging use of FFF as a pre-analytical step for mass-based identification and characterization of proteins and protein complexes in proteomics.     

Sedimentation field-flow-fractionation: emergence of a new cell separation methodology

Field flow fractionation (FFF) methods were conceptualised in the late 1960s by J.C Giddings. These techniques are particularly suited for the retention and separation of micron and sub-micron sized particles. Systematic technological development as well as methodological procedures were established to achieve separations over the last 30 years. The elution mechanism of micron sized species is now known as 'steric/hyperlayer'. Cells are micron sized particles of life science interest, in particular those living in suspension. The separation of cells according to differences in their biophysical characteristics is therefore possible using the FFF principle. In the first part of this report, characteristics of classical cell separation methodologies are recounted as well as the specific features of FFF. In the second part, a review of cell separations or purifications obtained with sedimentation FFF techniques is given and FFF trends in cell separation is developed.     

Size analysis of industrial carbon blacks by sedimentation and flow field-flow fractionation

Carbon black is one of the most useful particulate materials in the industrial field. Among the various physical properties of carbon black, size and size distribution are the most important properties to affect the quality of a final product. However, it is difficult to measure the exact particle size of carbon black since it suffers unavoidable interference from flocculation. In this study, the effects of various factors on the dispersion of industrial carbon blacks were investigated for the determination of size and size distribution of carbon black particles. Sedimentation and flow field-flow fractionations (FIFFF) were used to determine the size of carbon black, and their optimum analytical conditions were tested by changing surfactant, pH, ionic strength, and method of dispersion. The results showed that surfactant structure and its concentration played significant roles in dispersion stability. Carbon black was dispersed well with a nonionic surfactant with a pH of around 8 and an ionic strength of 0.003 M. The mean diameters measured from two types of FFF and photon correlation spectroscopy are in good agreement. This study demonstrates the potential of sedimentation and flow FFF for analyzing highly adsorptive industrial particles and guides for sample preparation.     

Trehalose Biosynthesis Enhancement for Six Yeast Strains Under Pressurized Culture

Six yeast strains of the commercial brewing yeasts CICC1391 and CICC1471, the commercial baker yeasts CICC1339 and CICC1447, and the commercial alcohol yeasts CICC1286 and CICC1291 have been cultured under 1.0 MPa of pressure with N₂ and CO₂ as pressure media. The concentration of intracellular trehalose and the activity of trehalose synthases complex have been measured. Also, the morphology changes of yeast cells have been observed by scanning electronic microscope. There was a positive correlation between the activity of trehalose synthase complex and the concentration of intracellular trehalose; and there was a negative correlation between the activity of trehalose synthase complex and the viability of yeast strains. Having been cultured for 3 h at high pressure of 1.0 MPa, the concentration of intracellular trehalose and the activity of trehalose synthases complex were improved by 50.1% to 116.4% and 45.2% to 219.1%, respectively, compared to those of atmospheric pressure culture. Under high pressure, many wrinkles appeared on the membrane surface of yeast cells. It has been found that yeasts are more sensitive to high pressure for having more and sharper wrinkles on their cell membranes.     

Separation of carbon nanotubes by frit inlet asymmetrical flow field-flow fractionation

Flow field-flow fractionation (flow FFF), a separation technique for particles and macromolecules, has been used to separate carbon nanotubes (CNT). The carbon nanotube ropes that were purified from a raw carbon nanotube mixture by acidic reflux followed by cross-flow filtration using a hollow fiber module were cut into shorter lengths by sonication under a concentrated acid mixture. The cut carbon nanotubes were separated by using a modified flow FFF channel system, frit inlet asymmetrical flow FFF (FI AFIFFF) channel, which was useful in the continuous flow operation during injection and separation. Carbon nanotubes, before and after the cutting process, were clearly distinguished by their retention profiles. The narrow volume fractions of CNT collected during flow FFF runs were confirmed by field emission scanning electron microscopy and Raman spectroscopy. Experimentally, it was found that retention of carbon nanotubes in flow FFF was dependent on the use of surfactant for CNT dispersion and for the carrier solution in flow FFF. In this work, the use of flow FFF for the size differentiation of carbon nanotubes in the process of preparation or purification was demonstrated.     

Application of a high-performance liquid chromatography fluorescence detector as a nephelometric turbidity detector following Field-Flow Fractionation to analyse size distributions of environmental colloids

A new operation mode for HPLC-type fluorescence detectors is presented and evaluated using synthetic and environmental particles in the colloidal size range. By applying identical wavelengths for excitation and emission a nephelometric turbidity or single angle light scattering detector is created which can be easily coupled to flow or sedimentation Field-Flow Fractionation (Flow FFF or Sed FFF) for the analysis of colloidal dispersions. The results are compared with standard UV-vis detection methods. Signals obtained are given as a function of particle size and selected detection wavelength. Conclusions can be drawn which affect the current practice of FFF but also for other techniques as groundwater sampling and laboratory column experiments when turbidity is measured in nephelometric mode and in small sample volumes or at low flow rates.     

HPLC of humic substances fractionated by Flow FFF

This communication reports a study of the effect of ionic strength and electrolyte composition on fractions, separately collected by flow FFF, of a mixture of humic substances. Reverse phase HPLC analysis of three early eluting fractions suggests that the components released by the column behave as organic acids. The baseline‐resolved peaks of the first two fractions, subject to higher retention in solutions of lower pH and/or higher polarity, substantiate this suggestion. The fraction with larger components, as measured by flow FFF, also appears to contain acidic species. Their retention level, however, may not be accurately modulated by varying the mobile phase properties as these species are either totally retained in acidic phases or released before the void peak at pH ≥ 4.2. Besides showing the effective separation achieved in the flow FFF channel, this study reveals the pronounced difference in the physicochemical properties of some components of a humic mixture even with very close particle size.     

D-Sorbitol Physical Properties Effects on Filaments Used by 3D Printing Process for Personalized Medicine

Fused filament fabrication (FFF) is a process used to manufacture oral forms adapted to the needs of patients. Polyethylene oxide (PEO) filaments were produced by hot melt extrusion (HME) to obtain a filament suitable for the production of amiodarone hydrochloride oral forms by FFF 3D printing. In order to produce personalized oral forms adapted to the patient characteristics, filaments used by FFF must be controlled in terms of mass homogeneity along filament. This work highlights the relation between filament mass homogeneity and its diameter. This is why the impact of filler excipients physical properties was studied. It has been showed that the particle’s size distribution of the filler can modify the filament diameter variability which has had an impact on the mass of oral forms produced by FFF. Through this work it was shown that D-Sorbitol from Carlo Erba allows to obtain a diameter variability of less than 2% due to its unique particle’s size distribution. Using the filament produced by HME and an innovating calibration method based on the filament length, it has been possible to carry out three dosages of 125 mg, 750 mg and 1000 mg by 3D printing with acceptable mass uniformity.