Asymmetrical Flow Field-flow Fractionation Research Articles

Automated On-Line Isolation and Fractionation Method for Subpopulations of Extracellular Vesicles.

Immunoaffinity chromatography (IAC) with selective antibodies immobilized on polymeric monolithic disk columns enables selective isolation of biomacromolecules from human plasma, while asymmetrical flow field-flow fractionation (AsFlFFF or AF4) can be used for further fractionation of relevant subpopulations of biomacromolecules (e.g., small dense low-density lipoproteins, exomeres, and exosomes) from the isolates. Here we describe how the isolation and fractionation of subpopulations of extracellular vesicles can be achieved without the presence of lipoproteins using on-line coupled IAC-AsFlFFF. With the developed methodology, it is possible to have fast, reliable, and reproducible automated isolation and fractionation of challenging biomacromolecules from human plasma with a high purity and high yields of subpopulations.

Phosphorus content in water extractable soil colloids over a 2000 years chronosequence of paddy-rice management in the Yangtze River Delta, China

Soil phosphorus (P) is usually hardly present in soluble forms as most of it is strongly bound to minerals. Therefore, transformations of soil colloidal P can play a key role in enhancing soil fertility. Here, we examined water extracts from a chronosequence of soils with 2000 years of paddy rice and 700 years of non-paddy cropping, and fractionated them into <1200 nm, <450 nm, and <3 kDa size fractions using sequential ultrafiltration. Asymmetric-flow field-flow fractionation (AF4) coupled to an organic carbon detector (OCD) and inductively coupled plasma mass spectrometry (ICP-MS) was used for organic carbon and elemental screening in the 3 kDa-450 nm colloidal size range. We found that P increased in all colloidal fractions both with long-term paddy rice and non-paddy managements. Furthermore, P in soil colloids of 3 kDa-450 nm size, as fractionated by AF4, peaked in three size ranges: nanoparticles (NP, ∼3 kDa-20 nm), fine colloids (FC, ∼20–225 nm), and medium-sized colloids (MC, ∼225–450 nm). The NP fraction originally contained mainly P and Ca for tidal wetland (TW), but turned into a complex of OC-Ca-P colloidal forms immediately after onset of arable management, with little temporal change thereafter. Fine colloidal P (FC-P) was detected only after > 50 years for the non-paddy and > 100 years under paddy management, associated with oxides, organic matter and fine clay fragments in the paddy soils and additionally Ca-bound P in the non-paddy ones, respectively. Increasing portions of medium-sized colloidal P appeared after 50 years paddy and non-paddy management, though not exceeding the concentrations of FC-P and with little changes during prolonged arable land use. Overall, after initial formation of FC-P during the first 50–100 years of management, longer term dynamics of colloidal P in these arable soils was fairly independent of paddy or non-paddy managements, but remained closely coupled to the increased presence or generation of fine colloidal clays, oxides and the associated P, thus potentially linking P fertility largely to the dynamics of fine colloids.

A Study on Aqueous Dispersing of Carbon Black Nanoparticles Surface-Coated with Styrene Maleic Acid (SMA) Copolymer.

Carbon black (CB) particles tend to aggregate in aqueous solutions, and finding an optimum dispersing condition (e.g., selection of the type of dispersant) is one of the important tasks in related industries. In the present study, three types of styrene maleic acid (SMA) copolymer dispersants were synthesized, labeled respectively 'SMA-1000', 'SMA-2000', and 'SMA-3000', which have 1, 2, and 3 styrene groups in their repeating units. Then, asymmetrical flow field-flow fractionation (AsFlFFF) was employed to measure the particle size distributions of the aqueous CB dispersions. For the particle size analysis of the CB dispersions, dynamic light scattering (DLS) showed relatively lower reproducibility than AsFlFFF. AsFlFFF showed that the use of SMA-3000 yielded a CB dispersion with the most uniform particle size distribution. When the SMA-3000 dispersant was used, the particle size tended to increase after 1 h of milling as the milling time increased, probably due to the re-agglomeration of the particles by excessive milling. The particle size distributions from AsFlFFF were consistent with the colorimetric observations. With the SMA-3000 dispersant, the lowest L∗ value was observed after 1 h of milling. The AsFlFFF and colorimetric analyses suggest that a stable CB dispersion can be obtained by either 3-h of milling with the SMA-2000 or 1-h of milling with the SMA-3000.

Simple, Reliable Protocol for High-Yield Solubilization of Seedless Amyloid-β Monomer.

Self-assembly of the amyloid-β (Aβ) peptide to form toxic oligomers and fibrils is a key causal event in the onset of Alzheimer's disease, and Aβ is the focus of intense research in neuroscience, biophysics, and structural biology aimed at therapeutic development. Due to its rapid self-assembly and extreme sensitivity to aggregation conditions, preparation of seedless, reproducible Aβ solutions is highly challenging, and there are serious ongoing issues with consistency in the literature. In this paper, we use a liquid-phase separation technique, asymmetric flow field-flow fractionation with multiangle light scattering (AF4-MALS), to develop and validate a simple, effective, economical method for re-solubilization and quality control of purified, lyophilized Aβ samples. Our findings were obtained with recombinant peptide but are physicochemical in nature and thus highly relevant to synthetic peptide. We show that much of the variability in the literature stems from the inability of overly mild solvent treatments to produce consistently monomeric preparations and is rectified by a protocol involving high-pH (>12) dissolution, sonication, and rapid freezing to prevent modification. Aβ treated in this manner is chemically stable, can be stored over long timescales at -80 °C, and exhibits remarkably consistent self-assembly behavior when returned to near-neutral pH. These preparations are highly monomeric, seedless, and do not require additional rounds of size exclusion, eliminating the need for this costly procedure and increasing the flexibility of use. We propose that our improved protocol is the simplest, fastest, and most effective way to solubilize Aβ from diverse sources for sensitive self-assembly and toxicity assays.

Tailor-Made Poly(vinylamine) via Purple LED-Activated RAFT Polymerization of N-vinylformamide.

Photo-iniferter reversible addition-fragmentation chain transfer (PI-RAFT) polymerization of N-vinylformamide (NVF) is demonstrated by using purple light. PNVFs with predetermined molar masses and narrow molar mass distributions are obtained. High RAFT chain-end fidelity is confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS), and chain extension experiment. To demonstrate the potential of this approach, an original poly(N-vinylpyrrolidone)-b-poly(N-vinylformamide) (PVP-b-PNVF) diblock copolymer is synthesized and characterized by aqueous size-exclusion chromatography (SEC), asymmetric flow field-flow fractionation (A4F), and 1 H diffusion-ordered spectroscopy nuclear magnetic resonance (1 H DOSY NMR). Finally, selective hydrolysis of PNVF block to corresponding pH-responsive poly(N-vinylpyrrolidone)-b-poly(N-vinylformamide) (PVP-b-PVAm) is performed.

Distinguishing nanoparticle drug release mechanisms by asymmetric flow field-flow fractionation.

This research demonstrates the development, application, and mechanistic value of a multi-detector asymmetric flow field-flow fractionation (AF4) approach to acquire size-resolved drug loading and release profiles from polymeric nanoparticles (NPs). AF4 was hyphenated with multiple online detectors, including dynamic and multi-angle light scattering for NP size and shape factor analysis, fluorescence for drug detection, and total organic carbon (TOC) to quantify the NPs and dissolved polymer in nanoformulations. The method was demonstrated on poly(lactic-co-glycolic acid) (PLGA) NPs loaded with coumarin 6 (C6) as a lipophilic drug surrogate. The bulk C6 release profile using AF4 was validated against conventional analysis of drug extracted from the NPs and complemented with high performance liquid chromatography - quadrupole time-of-flight (HPLC-QTOF) mass spectrometry analysis of oligomeric PLGA species. Interpretation of the bulk drug release profile was ambiguous, with several release models yielding reasonable fits. In contrast, the size-resolved release profiles from AF4 provided critical information to confidently establish the release mechanism. Specifically, the C6-loaded NPs exhibited size-independent release rate constants and no significant NP size or shape transformations, suggesting surface desorption rather than diffusion through the PLGA matrix or erosion. This conclusion was supported through comparative experimental evaluation of PLGA NPs carrying a fully entrapped drug, enrofloxacin, which showed size-dependent diffusive release, along with density functional theory (DFT) calculations indicating a higher adsorption affinity of C6 onto PLGA. In summary, the development of the size-resolved AF4 method and data analysis framework fulfills salient analytical gaps to determine drug localization and release mechanisms from nanomedicines.

Characterization of Oligomer Formation of Surfactant Protein-D (SP-D) Using AF4-MALLS.

Surfactant protein-S (SP-D) is a naturally occurring lung protein with the potential to treat pulmonary infections. A recombinant surfactant protein-D (SP-D) has been produced and was previously found to exist in multiple oligomeric states. Separation and characterization of interconverting oligomeric states of a protein can be difficult using chromatographic methods, so an alternative separation technique was employed for SPD to characterize the different association states that exist. Samples of SP-D were analyzed using asymmetrical flow field-flow fractionation (AF4) using UV and multi-angle laser light scattering (MALLS) detection. The AF4 method appears to be able to separate species as small as the monomer up to the dodecamer (the dominant species) to much larger species with a molar mass greater than 5 MDa. Consistent elution of four distinct peaks was observed after repeated injections. The largest species observed under the last peak (labeled as Peak 4) were termed "unstructured multimers" and were resolved fairly well from the other species. The AF4-MALLS data suggest that only a small fraction of Peak 4 truly corresponds to high molar mass unstructured multimers. All other peaks demonstrated significant molar mass homogeneity consistent with AFM results. AF4-MALLS technology appears to be a powerful analytical approach to characterize the complex and dynamic interplay among different protein oligomeric species of SP-D in an aqueous solution.

Soil colloidal particles in a subtropical savanna: Biogeochemical significance and influence of anthropogenic disturbances

Soil colloids (diameter < 1000 nm) are comprised mainly of clay minerals and organic matter, and play major roles in determining ion exchange capacity and in regulating key biogeochemical processes. Consequently, it is important to understand how soil colloids and their functions are influenced by land cover and anthropogenic disturbances. In grasslands, savannas, and other dryland ecosystems across the globe, woody plants are encroaching due to livestock grazing, fire suppression, elevated CO2 concentrations, and climate change. These major land cover changes could influence soil colloidal properties, with implications for soil C, N, and P cycles. We assessed how woody encroachment, livestock grazing, and fire interact to influence soil colloidal properties in a juniper-oak savanna. Surface soils (0–10 cm) from the southern Great Plains (Texas, USA) were collected from long-term treatments differing in grazing intensity (none, moderate, and heavy) and fire history. Within each treatment, soil samples were taken under grass, juniper, and oak canopies. Water dispersible soil colloids (WDC, d < 500 nm) were isolated and analyzed by asymmetric flow field-flow fractionation and their P species by liquid-state 31P-nuclear magnetic resonance spectroscopy (31P NMR). Soil beneath oak and juniper canopies had smaller WDC and elevated colloidal organic carbon (OC) and P concentrations, especially in nanocolloid (<30 nm) and fine colloid (30–160 nm) size fractions. Woody encroachment enriched Ca, Fe, Al, Si and Mg in WDC in the ungrazed control, but not in any of the other grazed or burned areas. Colloidal soil P mainly occurred as orthophosphate and orthophosphate diesters, and was present as OC-Ca-P complexes in fine and medium colloid fractions (30–500 nm), while P in the nanocolloid fraction (<30 nm) was in direct association with Ca. Moderate grazing did not affect the retention of colloidal P, while heavy grazing potentially increased the loss risk of colloidal P. Fire accelerated soil P loss from colloid fractions only in woody areas. Our findings highlight that woody encroachment strengthens the retention of OC and P by soil colloids, consequently increasing overall C and P pools in savanna soils.

Comparison of Different Isolation Methods for Plasma-Derived Extracellular Vesicles in Patients with Hyperlipidemia.

Extracellular vesicles are commonly found in human body fluids and can reflect current physiological conditions of human body and act as biomarkers of disease. The quality of isolated extracellular vesicles facilitates the early diagnosis of various diseases accompanied by hyperlipidemia. Nonetheless, there are no reports on which special methods are suitable for isolating extracellular vesicles from the plasma of patients with hyperlipidemia. Thus, this study compared three different research-based extracellular vesicle isolation approaches, namely ultracentrifugation (UC), polyethylene glycol (PEG) precipitation, and size exclusion chromatography (SEC), and determined which of them was the most effective method. We selected blood samples from 12 patients with clinically diagnosed hyperlipidemia and isolated plasma-derived extracellular vesicles using three methods. The morphology of the isolated extracellular vesicles was observed using transmission electron microscopy, while the concentration was detected by asymmetric flow field-flow fractionation and multi-angle light scattering. Marker proteins were identified by Western blotting, and protein composition was evaluated by silver staining. Both determined the contaminations in the extracellular vesicle samples. The results showed that the three methods can be successfully used for the isolation of extracellular vesicles. The extracellular vesicles isolated by UC were larger in size, and the yield was much lower. Although the yield of extracellular vesicles isolated by PEG precipitation was greatly improved, the contamination was increased. Of the three methods, only the SEC-isolated extracellular vesicles were characterized by high yield and low contamination. Therefore, our data suggested that the SEC was a more ideal method for isolating extracellular vesicles from the plasma of patients with hyperlipidemia.

Charge and zeta-potential distribution in starch modified with octenyl succinic anhydride (OSA) determined using electrical asymmetrical flow field-flow fractionation (EAF4)

Starch modified with octenyl succinic anhydride (OSA) is extensively used due to its emulsifying capacity. Modification with OSA renders the starch surface active but also anionic at intermediate pH. The amount of OSA that is grafted to the starch polymer backbone will influence the functionality. Likewise, it can be expected that the distribution of the substituents over the molar mass distribution will play an important role for the behavior and functionality. Electrical asymmetrical flow field-flow fractionation (EAF4) could provide charge-size dependent separation of samples with different charges. A major advantage is that zeta-potential and size/molar mass distribution can be simultaneously determined. The present study investigates if EAF4 can be used to determine the charge properties over molar mass (or size) distribution of barley starch modified with OSA. The results show that zeta potential and net charge could be estimated over the larger part of the OSA-starch molar mass distribution. Furthermore, the OSA substituents do not appear to be evenly distributed at or close to the “surface” of the polymers, which may have implications for the adsorption behavior and functionality of OSA-starch in emulsions. To the best of our knowledge, this is the first report on applying EAF4 for the characterization of biopolymers and the first time that zeta potential can be determined over the molar mass distribution.

Asymmetrical flow field-flow fractionation combined with ultrafiltration: A novel and high-efficiency approach for separation, purification, and characterization of Ganoderma lucidum polysaccharides

Ganoderma lucidum polysaccharide (GLP) has attracted increasing attention owing to its multiple pharmacological effects. However, the establishment of a structure-bioactivity relationship is possible only if highly purified and accurately characterized GLP is used. In this study, a combined technique based on asymmetrical flow field-flow fractionation (AF4), ultrafiltration, and the Sevag method was developed to improve the purification process of GLP. Deionized water was used throughout this study which avoids the dialysis desalting process. Furthermore, the molecular weight (Mw) and radius of gyration (Rg) of the purified GLP were characterized by AF4 coupled online ultraviolet–visible (UV), multiangle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI). The results demonstrated that after removal of the larger size proteins, one highly purified GLP fraction (polysaccharide content of 108.37 ± 0.45%) and its structural information (i.e., Mw, Rg, and apparent density) were obtained in a single run owing to a combination of mechanisms of ultrafiltration and AF4 separation. The results suggested that the method developed in this study for separation, purification, and characterization of GLP is highly efficient, which could help to better understand its structure-function relationships.

Variant APOL1 protein in plasma associates with larger particles in humans and mouse models of kidney injury.

BackgroundGenetic variants in apolipoprotein L1 (APOL1), a protein that protects humans from infection with African trypanosomes, explain a substantial proportion of the excess risk of chronic kidney disease affecting individuals with sub-Saharan ancestry. The mechanisms by which risk variants damage kidney cells remain incompletely understood. In preclinical models, APOL1 expressed in podocytes can lead to significant kidney injury. In humans, studies in kidney transplant suggest that the effects of APOL1 variants are predominantly driven by donor genotype. Less attention has been paid to a possible role for circulating APOL1 in kidney injury.MethodsUsing liquid chromatography-tandem mass spectrometry, the concentrations of APOL1 were measured in plasma and urine from participants in the Seattle Kidney Study. Asymmetric flow field-flow fractionation was used to evaluate the size of APOL1-containing lipoprotein particles in plasma. Transgenic mice that express wild-type or risk variant APOL1 from an albumin promoter were treated to cause kidney injury and evaluated for renal disease and pathology.ResultsIn human participants, urine concentrations of APOL1 were correlated with plasma concentrations and reduced kidney function. Risk variant APOL1 was enriched in larger particles. In mice, circulating risk variant APOL1-G1 promoted kidney damage and reduced podocyte density without renal expression of APOL1.ConclusionsThese results suggest that plasma APOL1 is dynamic and contributes to the progression of kidney disease in humans, which may have implications for treatment of APOL1-associated kidney disease and for kidney transplantation.

Investigation of native and aggregated therapeutic proteins in human plasma with asymmetrical flow field-flow fractionation and mass spectrometry

Physiochemical degradation of therapeutic proteins in vivo during plasma circulation after administration can have a detrimental effect on their efficacy and safety profile. During drug product development, in vivo animal studies are necessary to explore in vivo protein behaviour. However, these studies are very demanding and expensive, and the industry is working to decrease the number of in vivo studies. Consequently, there is considerable interest in the development of methods to pre-screen the behaviour of therapeutic proteins in vivo using in vitro analysis. In this work, asymmetrical flow field-flow fractionation (AF4) and liquid chromatography–mass spectrometry (LC-MS) were combined to develop a novel analytical methodology for predicting the behaviour of therapeutic proteins in vivo. The method was tested with two proteins, a monoclonal antibody and a serum albumin binding affibody. After incubation of the proteins in plasma, the method was successfully used to investigate and quantify serum albumin binding, analyse changes in monoclonal antibody size, and identify and quantify monoclonal antibody aggregates.Graphical abstract

Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles.

Extracellular vesicles (EVs) play important roles in cell-to-cell communications and carry high potential as markers targeted in disease diagnosis, prognosis, and therapeutic development. The main obstacles to EV study are their high heterogeneity; low amounts present in samples; and physical similarity to the abundant, interfering matrix components. Multiple rounds of separation and purification are often needed prior to EV characterization and function assessment. Herein, we report the offline coupling of asymmetrical flow field-flow fractionation (AF4) and capillary electrophoresis (CE) for EV analysis. While AF4 provides gentle and fast EV separation by size, CE resolves EVs from contaminants with similar sizes but different surface charges. Employing Western Blotting, ELISA, and SEM, we confirmed that intact EVs were eluted within a stable time window under the optimal AF4 and CE conditions. We also proved that EVs could be resolved from free proteins and high-density lipoproteins by AF4 and be further separated from the low-density lipoproteins co-eluted in AF4. The effectiveness of the coupled AF4-CE system in EV analysis was demonstrated by monitoring the changes in EV secretion from cells and by direct injection of human serum and detection of serum EVs. We believe that coupling AF4 and CE can provide rapid EV quantification in biological samples with much reduced matrix interference and be valuable for the study of total EVs and EV subpopulations produced by cells or present in clinical samples.

Analysis and purification of ssRNA and dsRNA molecules using asymmetrical flow field flow fractionation

Robust RNA purification and analysis methods are required to support the development of RNA vaccines and therapeutics as well as RNA interference -based crop protection solutions. Asymmetrical flow field-flow fractionation (AF4) is a gentle native purification method that applies liquid flows to separate sample components based on their hydrodynamic sizes. We recently showed that AF4 can be utilized to separate RNA molecules that are shorter than 110 nucleotides (nt), but the performance of AF4 in the analysis and purification of longer RNA molecules has not been previously evaluated. Here, we studied the performance of AF4 in separation of single-stranded (ss) and double-stranded (ds) RNA molecules in the size range of 75–6400 nt. In addition, we evaluated the power of AF4 coupling to different detectors, allowing separation to be combined with data collection on yield as well as molecular weight (MW) and size distribution. We show that AF4 method is applicable in RNA purification, quality control, and analytics, and results in good recoveries of ssRNA and dsRNA molecules. In addition, our results demonstrate the utility of AF4 multidetection platforms to study biophysical properties of long RNA molecules.

Investigation on the effects of drying methods on the structure and antioxidant activity of Tremella fuciformis polysaccharides using asymmetrical flow field‐flow fractionation

Polysaccharide is an important bioactive component in Tremella fuciformis (T. fuciformis). In this study, the effects of three drying methods (i.e., vacuum freeze-drying, microwave drying, and hot air drying) on the structure in terms of molecular weight (Mw) and radius of gyration, and conformation of T. fuciformis polysaccharide (TFP) were investigated by asymmetrical flow field-flow fractionation coupled online with multiangle light scattering and differential refractive index detectors. The physicochemical properties of TFPs were studied by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and differential scanning calorimetry. Furthermore, the structure-antioxidation relationship of TFPs was investigated. Results revealed that three drying methods did not significantly affect functional groups, crystal structure, and thermal property of TFPs but led to some differences in the morphology and Mw of TFPs. The TFP obtained by microwave drying had the highest DPPH radical scavenging activity, which could be attributed to low Mw. Novelty impact statement The three drying methods (i.e., vacuum freeze-drying, microwave drying, and hot air drying) studied in this work did not affect the functional groups and thermal properties of T. fuciformis polysaccharides (TFPs), but significantly affected the morphology of TFP granules and the Mw distribution of TFP molecules. TFP-M had the smallest Mw (1.36 ± 0.10 × 107 g/mol), which could be beneficial to its DPPH radical scavenging activity.

Implications of Free and Occluded Fine Colloids for Organic Matter Preservation in Arable Soils.

Colloidal organo-mineral associations contribute to soil organic matter (OM) preservation and mainly occur in two forms: (i) as water-dispersible colloids that are potentially mobile (free colloids) and (ii) as building units of soil microaggregates that are occluded inside them (occluded colloids). However, the way in which these two colloidal forms differ in terms of textural characteristics and chemical composition, together with the nature of their associated OM, remains unknown. To fill these knowledge gaps, free and occluded fine colloids <220 nm were isolated from arable soils with comparable organic carbon (Corg) but different clay contents. Free colloids were dispersed in water suspensions during wet-sieving, while occluded colloids were released from water-stable aggregates by sonication. The asymmetric flow field-flow fractionation analysis on the free and occluded colloids suggested that most of the 0.6-220 nm fine colloidal Corg was present in size fractions that showed high abundances of Si, Al, and Fe. The pyrolysis-field ionization mass spectrometry revealed that the free colloids were relatively rich in less decomposed plant-derived OM (i.e., lipids, suberin, and free fatty acids), whereas the occluded colloids generally contained more decomposed and microbial-derived OM (i.e., carbohydrates and amides). In addition, a higher thermal stability of OM in occluded colloids pointed to a higher resistance to further degradation and mineralization of OM in occluded colloids than that in free colloids. This study provides new insights into the characteristics of subsized fractions of fine colloidal organo-mineral associations in soils and explores the impacts of free versus occluded colloidal forms on the composition and stability of colloid-associated OM.

Application of Af4-Multidetection to Liraglutide in Its Formulation: Preserving and Representing Native Aggregation.

Aggregation is among the most critical parameters affecting the pharmacological and safety profile of peptide Active Pharmaceutical Ingredients (APIs). For this reason, it is of utmost importance to define the exact aggregation state of peptide drugs, particularly when the API is marketed as a ready-to-use solution. Consequently, appropriate non-destructive techniques able to replicate the peptide environment must be employed. In our work, we exploited Asymmetrical Flow Field-Flow Fractionation (AF4), connected to UV, dRI, fluorescence, and MALS detectors, to fully characterize the aggregation state of Liraglutide, a peptide API used for the treatment of diabetes type 2 and chronic obesity. In previous studies, Liraglutide was hypothesized to assemble into hexa-octamers in phosphate buffer, but no information on its behavior in the formulation medium was provided up to now. The method used allowed researchers to work using formulation as the mobile phase with excellent recoveries and LoQ/LoD, discerning between stable and degraded samples, and detecting, when present, aggregates up to 108 Da. The native state of Liraglutide was assessed and found to be an association into pentamers, with a non-spherical conformation. Combined to benchmark analyses, the sameness study was complete and descriptive, also giving insight on the aggregation process and covalent/non-covalent aggregate types.

Size-Resolved Fluorescence Underscores Negligible Interaction of Dissolved Organic Matter During Conservative Mixing in a Large Boreal River

Although river mixing occurs widely in nature, the corresponding evolution of dissolved organic matter (DOM) composition remains poorly understood. Here, surface water samples were collected at multiple transects in the lower Athabasca River (LAR) under base-flow conditions. Asymmetric flow field-flow fractionation (AF4) coupled to online excitation-emission measurements (EEMs) and parallel factor analysis (PARAFAC) were utilized to investigate the size distribution of fluorescent DOM components during river mixing and the corresponding variation in size-resolved fluorescence. The majority of fluorescent components occurred at 0.810 and 1.170 kDa, reflecting the small size of the DOM molecules with maximum fluorescence. The loadings of fluorescence normalized to absorbance at 254 nm (A254) were highest for most terrestrial humic-like components, followed by the microbial humic-like component, and the protein-like components. Differences in size-resolved fluorescence were observed between DOM in humic-rich tributaries and in the mainstem of the LAR upstream of tributary inputs. The trend of variations in the A254-normalized PARAFAC loadings of terrestrial humic-like components also illustrates conservative mixing of aromatic-rich terrestrial DOM across size fractions in the LAR. From a molecular point of view, the mixing of fluorescent DOM occurred linearly and simultaneously across sizes without any evidence of aggregation, sedimentation, or changes in the fluorescence or concentration of any size fraction over the &amp;gt;60 km required for complete mixing of the river and its tributaries. Overall, this study provides insights into the size characteristics of fluorescent components of DOM and their conservative mixing behavior in large boreal rivers.

Asymmetrical flow field-flow fractionation combined with liquid chromatography enables rapid, quantitative, and structurally informative detection of resistant starch

Resistant starch (RS) is one kind of biomacromolecules with prebiotic effects. Nowadays, it is still a challenge to characterize structure and content of RS over entire molecular weight (Mw) distribution. In this study, a combined technique of asymmetrical flow field-flow fractionation (AF4) and liquid chromatography (LC) for the accurate structure characterization and quantitative analysis of RS was developed. AF4 coupled online with multiangle light scatting (MALS) and differential refractive index (dRI) detectors (AF4-MALS-dRI) was used to characterize the structural information (radius of gyration and Mw) and content of RS with Mw larger than 5 kDa. Meanwhile, RS was characterized by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The glucose content in the digested starch solution was quantitatively determined using LC. The results demonstrated that AF4-LC is a simple and rapid method for structurally informative detection and quantitative analysis of RS over entire Mw distribution.