Hollow-fiber Flow Field-flow Fractionation Research Articles

On-line coupling of hollow-fiber flow field-flow fractionation and depolarized multi-angle static light scattering (HF5/D-MALS). Proof of principle

Introduced here is the on-line coupling of hollow-fiber flow field-flow fractionation (HF5) to depolarized multi-angle static light scattering (D-MALS). HF5 is a size-based separation alternative to size-exclusion and hydrodynamic chromatography and asymmetric flow field-flow fractionation. HF5 can separate larger sizes than its chromatographic counterparts and provides several advantages over its fractionation counterpart, including reduced sample consumption and greater ease of operation. D-MALS is a variant of MALS in which the depolarized scattering from the analyte solution is measured at a variety of angles simultaneously. Measurements of depolarized scattering have previously been employed in studying the optical properties of solutions or suspensions, to determine the length of rod-like analytes, and to gain increased accuracy in the determination of analyte molar mass. The coupling HF5/D-MALS allows for the depolarization ratio of a solution or suspension to be measured continuously across the fractogram. This is demonstrated here for a Teflon latex the size range of which extends beyond that accessible to commercial size-exclusion columns. The results presented provide the first reported on-line HF5/D-MALS coupling, showing the feasibility of the technique as well as its realized potential for providing continuous depolarization measurements, inter alia.

Emerging Microfluidic Tools for Simultaneous Exosomes and Cargo Biosensing in Liquid Biopsy: New Integrated Miniaturized FFF-Assisted Approach for Colon Cancer Diagnosis

The early-stage diagnosis of cancer is a crucial clinical need. The inadequacies of surgery tissue biopsy have prompted a transition to a less invasive profiling of molecular biomarkers from biofluids, known as liquid biopsy. Exosomes are phospholipid bilayer vesicles present in many biofluids with a biologically active cargo, being responsible for cell-to-cell communication in biological systems. An increase in their excretion and changes in their cargo are potential diagnostic biomarkers for an array of diseases, including cancer, and they constitute a promising analyte for liquid biopsy. The number of exosomes released, the morphological properties, the membrane composition, and their content are highly related to the physiological and pathological states. The main analytical challenge to establishing liquid biopsy in clinical practice is the development of biosensors able to detect intact exosomes concentration and simultaneously analyze specific membrane biomarkers and those contained in their cargo. Before analysis, exosomes also need to be isolated from biological fluids. Microfluidic systems can address several issues present in conventional methods (i.e., ultracentrifugation, size-exclusion chromatography, ultrafiltration, and immunoaffinity capture), which are time-consuming and require a relatively high amount of sample; in addition, they can be easily integrated with biosensing systems. A critical review of emerging microfluidic-based devices for integrated biosensing approaches and following the major analytical need for accurate diagnostics is presented here. The design of a new miniaturized biosensing system is also reported. A device based on hollow-fiber flow field-flow fractionation followed by luminescence-based immunoassay is applied to isolate intact exosomes and characterize their cargo as a proof of concept for colon cancer diagnosis.

Characterization of nano-minerals and nanoparticles in supergene rare earth element mineralization related to chemical weathering of granites

AbstractIon adsorption-type rare earth element (REE) ore deposits in South China are a major source of heavy rare earth elements (HREE) around the world, which are of considerable economic and strategic significance. In these ores, REE is enriched in the clay minerals, specifically kaolinite and halloysite, which are derived from their parent granitoid by the weathering process. However, the mechanisms of supergene REE mineralization remain unclear. We investigated the nature and origin of supergene REE mineralization, based on a nanoscale study of a typical REE-mineralized granite regolith profile (ΣREEmax = 1201 ppm) in the Dazhou super-large, ion adsorption-type REE deposit, Guangxi Province, South China. Bulk mineralogical and geochemical analyses, coupled with novel nano-characterization techniques [i.e., hollow fiber flow field-flow fractionation inductively coupled plasma-mass spectrometry (HF5-ICP-MS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM)], were used to determine the nature of the nano-minerals and nanoparticles in the regolith samples. X-ray diffraction and SEM-EDS analyses revealed that ion-adsorption clay minerals are dominated by platy-shaped kaolinite and rod-like halloysite (10 and 7 Å) within the regolith. The average clay mineral contents decreased from 38 to 15% from the fully weathered horizon to the semi-weathered horizon, whereas the proportion of halloysite increased in the clay mineral fraction in the deep horizons. The REE-bearing nanoparticles consist predominantly of macromolecules of organic matter (2–5 nm) and clay minerals (5–40 and 40–80 nm) according to the HF5-ICP-MS analysis. There is a close association between REE and Al contents in particles with sizes of 5–40 nm in the semi-weathered horizons and 40–80 nm in the highly weathered horizons, which indicates that nanoscale clay minerals (halloysite and kaolinite, respectively) are important REE carriers. In addition, nanoscale secondary REE mineral phases, including oxide, silicate, and phosphate, were identified by the SEM and TEM observations. These phases are typically adsorbed onto the surfaces of clay minerals, specifically rod-like halloysite, but have different occurrences in the regolith profile. Cesium-oxide (cerianite) and Ce-silicate (cerite) occur mainly in the upper horizon of the regolith profile, whereas low-crystallinity REE phosphates [rhabdophane-(La)] occur mainly in the lower horizon of the profile. Our results indicate that nano-minerals and nanoparticles affect REE enrichment and fractionation during granite weathering. Migration and accumulation of REE-bearing nano-minerals were caused by leaching and neoformation of REE-bearing nano-minerals during secondary precipitation. These processes contribute to the formation of supergene REE mineralization in granite regolith.

Tracking Heme-Protein Interactions in Healthy and Pathological Human Serum in Native Conditions by Miniaturized FFF-Multidetection

The interaction of heme with blood serum proteins plays an important role in many physiological and pathological processes involving enzyme activity, gene expression and cell proliferation. The mechanisms underlying these interactions are; however, not yet fully understood. New analytical methods able to investigate protein-heme binding in native, biologically representative conditions are thus required. In this work, we present a method based on miniaturized, hollow-fiber flow field-flow fractionation with multiple spectrophotometric and light-scattering detection for size separation of high-abundance serum proteins and selective detection of heme-bound subpopulations. Heme is found to mainly interact with serum albumin, whereas a low amount also binds to other proteins such as IgM. The ability to bind heme in physiological conditions is also investigated for individual serum proteins. IgG is found unable to bind heme at clinically relevant concentrations. The proposed method allows separation, quantitation, and mass/size characterization of serum high-abundance proteins, providing information of heme-protein complex stability and preferred heme-clearing pathways. The same approach could be in perspective extended to the investigation of specific heme-antibody binding, and to further studies involving other molecules of pharmaceutical/clinical interest.

Application of non-stationary phase separation hyphenated with inductively coupled plasma mass spectrometry in the analysis of trace metal-containing nanoparticles in the environment

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布、化学组成等信息,在金属纳米颗粒的分离检测方面表现出极大的潜能。但这一联用技术尚无法获得金属纳米颗粒物的颗粒数浓度和单个颗粒的元素信息,难以判断金属纳米颗粒涂层厚度、纯度以及颗粒的均相/异相团聚行为等。新兴的单颗粒-电感耦合等离子体质谱(SP-ICP-MS)与无固定相分离技术的在线联用,可以获得金属纳米颗粒的流体动力学粒径、元素质量计算粒径和颗粒数浓度等信息,进而弥补无固定相分离与ICP-MS在线联用技术的不足。该文介绍了流体动力色谱、毛细管电泳和场流分离3种常用无固定相分离技术的分离机制和适用检测器,着重综述了无固定相分离技术与ICP-MS/SP-ICP-MS在线联用技术的特点及其在环境金属纳米颗粒分析中的应用。关于场流分离,主要介绍了可以与ICP-MS联用的沉降场流分离和流场流分离。该文还对流体动力色谱、毛细管电泳和流场流分离与ICP-MS在线联用技术的特点进行了比较。最后,该文对无固定相分离技术与ICP-MS/SP-ICP-MS在线联用技术的发展提出了展望。

Tracking the dissolution behavior of zinc oxide nanoparticles in skimmed milk powder solutions

Zinc oxide nanoparticles (ZnO NPs) are recently recommended as food additives owing to their outstanding nutritive function. Therefore, understanding their comprehensive information and stability in food samples is highly necessitated. However, the characterization of ZnO NPs in the complex food matrices remains a great challenge, limiting an in-depth understanding of their transformation during food storage. In this study, the hollow fiber flow field-flow fractionation was combined with UV–Vis absorption spectroscopy and inductively coupled plasma optical emission spectroscopy to assess the dissolution behaviors of ZnO NPs in skimmed milk powder solutions by monitoring the changes in the residual ZnO NPs and the amount of dissolved Zn(II) ions. The simultaneous characterization of these two Zn species in skimmed milk powder solutions was achieved without the need for tedious sample pretreatments, and the dissolution of ZnO NPs in skimmed milk powder solutions had time- and temperature-dependent behaviors.

An ultracentrifugation – hollow-fiber flow field-flow fractionation orthogonal approach for the purification and mapping of extracellular vesicle subtypes

In the course of their life span, cells release a multitude of different vesicles in the extracellular matrix (EVs), constitutively and/or upon stimulation, carrying signals either inside or on their membrane for intercellular communication. As a natural delivery tool, EVs present many desirable advantages, such as biocompatibility and low toxicity. However, due to the complex biogenesis of EVs and their high heterogeneity in size distribution and composition, the characterization and quantification of EVs and their subpopulations still represents an enticing analytical challenge.Centrifugation methods allow to obtain different subpopulations in an easy way from cell culture conditioned medium and biological fluids including plasma, amniotic fluid and urine, but they still present some drawbacks and limitations. An unsatisfactory isolation can limit their downstream analysis and lead to wrong conclusions regarding biological activities. Isolation and characterization of biologically relevant nanoparticles like EVs is crucial to investigate specific molecular and signaling patterns and requires new combined approaches.Our work was focused on HF5 (miniaturized, hollow-fiber flow field-flow fractionation), and its hyphenation to ultracentrifugation techniques, which are the most assessed techniques for vesicle isolation. We exploited model samples obtained from culture medium of murine myoblasts (C2C12), known to release different subsets of membrane-derived vesicles.Large and small EVs (LEVs and SEVs) were isolated by differential ultracentrifugation (UC). Through an HF5 method employing UV, fluorescence and multi-angle laser scattering as detectors, we characterized these subpopulations in terms of size, abundance and DNA/protein content; moreover, we showed that microvesicles tend to hyper-aggregate and partially release nucleic matter. The quali-quantitative information we obtained from the fractographic profiles was improved with respect to Nano Tracking Analysis (NTA) estimation.The SEV population was then further separated using density gradient centrifugation (DGC), and four fractions were submitted again to HF5-multidetection. This technique is based on a fully orthogonal principle, since F4 does not separate by density, and provided uncorrelated information for each of the fractions processed. The “second dimension” achieved with HF5 showed good promise in sorting particles with both different size and content, and allowed to identify the presence of fibrilloid nucleic matter. This analytical bidimensional approach proved to be effective for the characterization of highly complex biological samples such as mixtures of EVs and could provide purified fractions for further biological characterization.

Perspectives on protein biopolymers: miniaturized flow field-flow fractionation-assisted characterization of a single-cysteine mutated phaseolin expressed in transplastomic tobacco plants

The development of plant-based protein polymers to employ in biofilm production represents the promising intersection between material science and sustainability, and allows to obtain biodegradable materials that also possess excellent physicochemical properties. A possible candidate for protein biopolymer production is phaseolin, a storage protein highly abundant in P Vulgaris beans. We previously showed that transformed tobacco chloroplasts could be employed to express a mutated phaseolin carrying a signal peptide (directing it into the thylakoids) also enriched of a cysteine residue added to its C-terminal region. This modification allows for the formation of inter-chain disulfide bonds, as we previously demonstrated, and should promote polymerization.To verify the effect of the peptide modification and to quantify polymer formation, we employed hollow-fiber flow field-flow fractionation coupled to UV and multi-angle laser scattering detection (HF5-UV-MALS): HF5 allows for the selective size-based separation of phaseolin species, whereas MALS calculates molar mass and conformation state of each population. With the use of two different HF5 separation methods we first observed the native state of P.Vulgaris phaseolin, mainly assembled into trimers, and compared it to mutated phaseolin (P*) which instead resulted highly aggregated. Then we further characterized P* using a second separation method, discriminating between two and distinct high-molecular weight (HMW) species, one averaging 0.8 × 106 Da and the second reaching the tens of million Da. Insight on the conformation of these HMW species was offered from their conformation plots, which confirmed the positive impact of the Cys modification on polymerization.

On-line determination of soluble Zn content and size of the residual fraction in PM2.5 incubated in various aqueous media

Transition metals in airborne particulate matter, especially those with aerodynamic diameters no more than 2.5 μm (PM2.5), have attracted considerable attention due to their potential environmental and human health risks. However, determination of these potential risks requires comprehensive knowledge of their dissolution behavior and residual size in aqueous media. Herein, we describe an analytical method for on-line determination of the soluble fraction of Zn as a model transition metal and the size of residual PM2.5 using hollow fiber flow field-flow fractionation (HF5) coupled with UV–vis absorption spectroscopy and inductively coupled plasma optical emission spectroscopy. HF5 was directly applied on the incubated samples in pure water (PW), simulated natural water (SNW), and simulated lung fluid (SLF) due to its efficient in-line filtration and excellent fractionation resolution. Firstly, the potential of the proposed method (under optimized conditions) for size characterization was assessed against commercial silica microparticles, and results in good agreement with manufacturer and scanning electron microscopy values were obtained. The accuracy of quantification of soluble Zn in various media was then validated using a standard reference material in terms of satisfactory recoveries compared with the reference values. For the real PM2.5 samples collected from different sites in Beijing, China, the soluble Zn percentages in PW, SNW, and SLF were within 15.4–16.7%, 10.6–12.7%, and 43.1–46.9%, respectively, with the amount of particles smaller than ~10 nm released from PM2.5 increasing in the order of SNW < PW < SLF. The proposed HF5-based method provides a powerful and efficient tool for the quantification of soluble transition metal fractions and size characterization of residual particles with reduced analysis times, thus possessing great promise in real-time tracking of the transformation of PM2.5 in environmental and physiological media and in risk assessment.

A new approach for the separation, characterization and testing of potential prionoid protein aggregates through hollow-fiber flow field-flow fractionation and multi-angle light scattering

Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior.The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension.In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests.After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.

Impact of Ionic Strength of Carrier Liquid on Recovery in Flow Field-Flow Fractionation

Asymmetrical flow field-flow fractionation (AF4) and hollow-fiber flow field-flow fractionation (HF5) are techniques widely used in analytical, industrial and biological analyses. The main problem in all AF4 and HF5 analyses is sample loss due to analyte–membrane interactions. In this work the impact of liquid carrier composition on latex nanoparticles (NPs) separation in water and two different concentrations of NH4NO3 was studied. In AF4, a constant trend of decreasing the size of 60 and 121.9 nm particles induced by the ionic strength of the carrier liquid has been observed. In contrast, an agglomeration effect of the biggest 356 nm particles was observed when increasing ionic strength, which induced a significant drop of recovery to 35%. H5F provides better resolution and intensified peaks of NPs, but careful optimisation of system parameters is mandatory to obtain good separation.

Application of hollow fiber flow field-flow fractionation with UV–Vis detection in the rapid characterization and preparation of poly(vinyl acetate) nanoemulsions

Poly(vinyl acetate) (PVAc) nanoemulsions have wide applications in the construction and adhesives industries, with their unique properties being closely dependent on the PVAc nanoparticle (NP) size. Effective and robust analytical methods are needed to accurately characterize the NP size according to the specific requirements to satisfy quality controls and scale production demands. Herein, hollow fiber flow field-flow fractionation (HF5) with UV–Vis detection, which features instrumental simplicity, convenient operation, and low running costs, was proposed for the real-time size characterization of PVAc NPs. The results obtained by the proposed method were in good agreement with the size values obtained by TEM, thereby demonstrating the applicability and reliability of the HF5-based method for PVAc NP size characterization without tedious sample preparation requirements. In order to assess the parameters dominating PVAc NP size, the variables of emulsifier concentration, composite emulsifier ratio, reaction temperature, and stirring rate were investigated in detail. The results proved that the emulsifier component and string rates were the key parameters controlling particle size. Furthermore, NP growth processes were tracked by HF5 over 8h, clearly indicating a gradual increase in PVAc NP size with time. The results firmly confirmed that HF5 has great potential in NP size control applications during the production and product quality evaluation of PVAc NPs.

Characterization of therapeutic protein AvidinOX by an integrated analytical approach.

AvidinOX, the oxidized derivative of Avidin, is a chemically modified glycoprotein, being currently under clinical investigation for targeted delivery of radioactive biotin to inoperable tumors. AvidinOX is produced by 4-hydroxyazobenzene-2-carboxylic acid (HABA)-assisted sodium periodate oxidation of Avidin. The peculiar property of the periodate-generated glycol-split carbohydrate moieties to form Schiff's bases with amino groups of the tissue proteins allows to achieve a tissue half-life of 2weeks compared to 2h of native Avidin. Carbohydrate oxidation, along with possible minor amino acid modifications, introduces additional microheterogeneity in the glycoprotein structure, making its characterization even more demanding than for native glycoproteins. Aiming at the elucidation of the effects of oxidation conditions on the AvidinOX protein backbone and sugars, this microheterogeneous glycoprotein derivative was characterized for the first time using a combination of different analytical methods, including colorimetric methods, mass spectrometry, hollow-fiber flow field-flow fractionation with UV and multi-angle laser scattering detection (HF5-UV-MALS), and NMR. The proposed integrated approach reveals structural features of AvidinOX relevant for its biological activity, e.g., oxidized sites within both carbohydrate moieties and protein backbone and conformational stability, and will be considered as an analytical tool for AvidinOX industrial preparations. It is worth noting that this study enriches also the structural data of native Avidin published up-to-date (e.g., glycan structure and distribution, peptide fingerprint, etc.). Graphical abstract Scheme of phenylacetic hydrazide/MALDI-TOF approach for quantification of aldehydes in AvidinOX based on the determination of the number of hydrazone adducts between hydrazide reagent and aldehyde groups of protein.

Tracking the Transformation of Nanoparticulate and Ionic Silver at Environmentally Relevant Concentration Levels by Hollow Fiber Flow Field-Flow Fractionation Coupled to ICPMS.

It is a great challenge to monitor the physical and chemical transformation of nanoparticles at environmentally relevant concentration levels, mainly because the commonly used techniques like dynamic light scattering and transmission electron microscopy are unable to characterize and quantify trace level nanoparticles in complex matrices. Herein, we demonstrate the on-line coupled system of hollow fiber flow field-flow fractionation (HF5), minicolumn concentration, and inductively coupled plasma mass spectrometry (ICPMS) detection as an efficient approach to study the aggregation and chemical transformation of silver nanoparticles (AgNPs) and ionic Ag species in the aqueous environment at ng/mL levels. Taking advantage of the in-line dialysis of HF5, the selective capture of Ag(I) species by the resin in minicolumn, and the high selectivity and sensitivity of ICPMS detection, we recorded the aggregation of 10 ng/mL AgNPs in complex matrices (e.g., NOM, Na+/Ca2+), revealing an interesting tiny AgNPs formation process of photoreduction of trace level Ag(I) that is different from larger AgNPs generated at high concentration of Ag(I) by accurate characterization and respectively identifying and quantifying new thiol-complexed Ag(I) and residual Ag(I) in the intertransformation of Ag(I) and AgNPs in domestic wastewater by simultaneously detecting the S and Ag signals via ICPMS.

Specific refractive index increment (∂n/∂c) of polymers at 660 nm and 690 nm.

The specific refractive index increment (∂n/∂c) is an essential datum for the accurate quantitation of molar mass averages and distributions (inter alia) of macromolecules when refractometry, static light scattering, and/or viscometry detection are coupled on-line to size-based separation techniques. The latter include methods such as size-exclusion and hydrodynamic chromatography, and asymmetric and hollow-fiber flow field-flow fractionation. The ∂n/∂c is also needed for accurate determination of the weight-average molar mass of polymers by off-line, batch-mode multi-angle static light scattering. However, not only does ∂n/∂c differ among chemical species, it also depends on experimental conditions such as solvent, temperature, and wavelength. For the last seventeen years, the author's laboratories have measured the ∂n/∂c of a variety of natural and synthetic polymers, at both 690 nm and, more recently, 660 nm, under a variety of solvent and temperature conditions. In all cases, this has been done by off-line, batch-mode differential refractometry, not by assuming 100% analyte column recovery and 100% accurate peak integration. Results of these determinations are presented here, along with the relevant experimental data.

Hollow-fiber flow field-flow fractionation and multi-angle light scattering as a new analytical solution for quality control in pharmaceutical nanotechnology

The rapid development of nanoproducts in pharmaceutical field highlights the need for robust analytical methods to ensure their quality and stability. Nanoparticles of different nature and composition (NPs) are employed for many purposes, such as the improvement of drug solubility/bioavailability and the controlled delivery of drugs. Among NPs features, particle size distribution (PSD) plays a fundamental role in determining NPs properties. Nevertheless the high development of different NPs, authorities such as the FDA and the European Union highlight the lack of robust characterization methods and quality control for nanomaterials.Among the techniques for the size-characterization of particles, Field-Flow Fractionation (FFF) represents a competitive choice: due to the absence of a stationary phase, the separation mechanism is gentle with total maintenance of the native properties of the analytes. In this paper the microcolumn variant of FFF, the Hollow-Fiber Flow FFF (HF5), is coupled on-line with Multi-Angle Light Scattering (MALS) for the development of methods for the characterization of NPs as quality control in new pharmaceutical field. The HF5-MALS was applied to the size characterization in different preservation conditions of silver polyvinylpyrrolidone-stabilized NPs (AgPVP) used for their antimicrobial activity. The ratio of gyration and hydrodynamic radii of AgPVP was evaluated for fresh and aged NPs, suggesting in this case aggregation rearrangement. The influence of different PVP coating and dilution factor was also studied. Finally the metal ion release was determined in relationship to these shape modifications. The HF5-MALS method is robust and reproducible and it can be considered as an important tool for the development of analytical platform for quality control of NPs.

Size determination and quantification of engineered cerium oxide nanoparticles by flow field-flow fractionation coupled to inductively coupled plasma mass spectrometry

Facing the lack of studies on characterization and quantification of cerium oxide nanoparticles (CeO2 NPs), whose consumption and release is greatly increasing, this work proposes a method for their sizing and quantification by Flow Field-flow Fractionation (FFFF) coupled to Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Two modalities of FFFF (Asymmetric Flow- and Hollow Fiber-Flow Field Flow Fractionation, AF4 and HF5, respectively) are compared, and their advantages and limitations discussed. Experimental conditions (carrier composition, pH, ionic strength, crossflow and carrier flow rates) are studied in detail in terms of NP separation, recovery, and repeatability. Size characterization of CeO2 NPs was addressed by different approaches. In the absence of feasible size standards of CeO2 NPs, suspensions of Ag, Au, and SiO2 NPs of known size were investigated. Ag and Au NPs failed to show a comparable behavior to that of the CeO2 NPs, whereas the use of SiO2 NPs provided size estimations in agreement to those predicted by the theory. The latter approach was thus used for characterizing the size of CeO2 NPs in a commercial suspension. Results were in adequate concordance with those achieved by transmission electron microscopy, X-ray diffraction and dynamic light scattering. The quantification of CeO2 NPs in the commercial suspension by AF4-ICP-MS required the use of a CeO2 NPs standards, since the use of ionic cerium resulted in low recoveries (99±9% vs. 73±7%, respectively). A limit of detection of 0.9μgL−1 CeO2 corresponding to a number concentration of 1.8×1012L−1 for NPs of 5nm was achieved for an injection volume of 100μL.

Separation of silver nanoparticles by hollow fiber flow field-flow fractionation: Addition of tannic acid into carrier liquid as a modifier

A homemade hollow fiber flow-field fractionation (Hf-FlFFF) coupled with inductively coupled plasma mass spectrometry (ICP-MS) was set-up for silver nanoparticles (AgNPs) separation by using polysulfone hollow fiber membrane (30,000 MW cutoff) as a separation channel. Tannic acid and citrate stabilized AgNPs were synthesized and introduced into Hf-FlFFF. The effects of carrier liquid and stabilizing agent on retention behavior of AgNPs were investigated. Different elution behaviors were observed as follows: with 0.02% (w/v) FL-70, all of AgNPs were eluted from Hf-FlFFF but differences in retention behaviors were observed for AgNPs with tannic acid and citrate stabilizing agents; and with 30mM TRIS buffer, only tannic acid stabilized AgNPs were eluted from Hf-FlFFF, whereas citrate stabilized AgNPs were not eluted. In this work, tannic acid addition into carrier liquid was proposed to modify the surface of AgNPs and the surface of the membrane, and thereby adjusting the retention behaviors of AgNPs. Various concentrations of tannic acid were added into FL-70 and TRIS buffer. With the use of 0.1mM tannic acid in 30mM TRIS buffer as the carrier liquid, retention behaviors of both tannic acid stabilized- and citrate stabilized-AgNPs were similar and with similar fractionation recovery.

Toward full spectrum speciation of silver nanoparticles and ionic silver by on-line coupling of hollow fiber flow field-flow fractionation and minicolumn concentration with multiple detectors.

The intertransformation of silver nanoparticles (AgNPs) and ionic silver (Ag(I)) in the environment determines their transport, uptake, and toxicity, demanding methods to simultaneously separate and quantify AgNPs and Ag(I). For the first time, hollow fiber flow field-flow fractionation (HF5) and minicolumn concentration were on-line coupled together with multiple detectors (including UV-vis spectrometry, dynamic light scattering, and inductively coupled plasma mass spectrometry) for full spectrum separation, characterization, and quantification of various Ag(I) species (i.e., free Ag(I), weak and strong Ag(I) complexes) and differently sized AgNPs. While HF5 was employed for filtration and fractionation of AgNPs (>2 nm), the minicolumn packed with Amberlite IR120 resin functioned to trap free Ag(I) or weak Ag(I) complexes coming from the radial flow of HF5 together with the strong Ag(I) complexes and tiny AgNPs (<2 nm), which were further discriminated in a second run of focusing by oxidizing >90% of tiny AgNPs to free Ag(I) and trapped in the minicolumn. The excellent performance was verified by the good agreement of the characterization results of AgNPs determined by this method with that by transmission electron microscopy, and the satisfactory recoveries (70.7-108%) for seven Ag species, including Ag(I), the adduct of Ag(I) and cysteine, and five AgNPs with nominal diameters of 1.4 nm, 10 nm, 20 nm, 40 nm, and 60 nm in surface water samples.

Top-down and bottom-up lipidomic analysis of rabbit lipoproteins under different metabolic conditions using flow field-flow fractionation, nanoflow liquid chromatography and mass spectrometry

This study demonstrated the performances of top-down and bottom-up approaches in lipidomic analysis of lipoproteins from rabbits raised under different metabolic conditions: healthy controls, carrageenan-induced inflammation, dehydration, high cholesterol (HC) diet, and highest cholesterol diet with inflammation (HCI). In the bottom-up approach, the high density lipoproteins (HDL) and the low density lipoproteins (LDL) were size-sorted and collected on a semi-preparative scale using a multiplexed hollow fiber flow field-flow fractionation (MxHF5), followed by nanoflow liquid chromatography-ESI-MS/MS (nLC-ESI-MS/MS) analysis of the lipids extracted from each lipoprotein fraction. In the top-down method, size-fractionated lipoproteins were directly infused to MS for quantitative analysis of targeted lipids using chip-type asymmetrical flow field-flow fractionation-electrospray ionization-tandem mass spectrometry (cAF4-ESI-MS/MS) in selected reaction monitoring (SRM) mode. The comprehensive bottom-up analysis yielded 122 and 104 lipids from HDL and LDL, respectively. Rabbits within the HC and HCI groups had lipid patterns that contrasted most substantially from those of controls, suggesting that HC diet significantly alters the lipid composition of lipoproteins. Among the identified lipids, 20 lipid species that exhibited large differences (>10-fold) were selected as targets for the top-down quantitative analysis in order to compare the results with those from the bottom-up method. Statistical comparison of the results from the two methods revealed that the results were not significantly different for most of the selected species, except for those species with only small differences in concentration between groups. The current study demonstrated that top-down lipid analysis using cAF4-ESI-MS/MS is a powerful high-speed analytical platform for targeted lipidomic analysis that does not require the extraction of lipids from blood samples.