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Poly(N‐vinylcaprolactam) (PVCL), a salt responsive ligand, was grafted from electrospun cellulose nanofiber membranes by atom transfer radical polymerization (ATRP). The membranes were used for hydrophobic interaction chromatography. The modified PVCL‐cellulose membranes were used to separate BSA dimers from monomers as well as monoclonal antibody (mAb) aggregates. Fast protein liquid chromatography (FPLC) was used for protein separation and fraction collection. High performance liquid chromatography with a size exclusion column (HPLC‐SEC) analysis confirmed the separation of monomers and aggregates from both BSA and the mAb. The results suggest that use of a responsive ligand that changes its conformation with ionic strength could improve the performance of hydrophobic interaction chromatography enabling the separation of compounds based on slight differences in hydrophobicity. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are cell derived membranous nanoparticles. EVs are important mediators of cell–cell communication via the transfer of bioactive content and as such they are being investigated for disease diagnostics as biomarkers and for potential therapeutic cargo delivery to recipient cells. However, existing methods for isolating EVs from biological samples suffer from challenges related to co‐isolation of unwanted materials such as proteins, nucleic acids, and lipoproteins. In the pursuit of improved EV isolation techniques, we introduce multimodal flowthrough chromatography (MFC) as a scalable alternative to size exclusion chromatography (SEC). The use of MFC offers significant advantages for purifying EVs, resulting in enhanced yields and increased purity with respect to protein and nucleic acid co‐isolates from conditioned 3D cell culture media. Compared to SEC, significantly higher EV yields with similar purity and preserved functionality were also obtained with MFC in 2D cell cultures. Additionally, MFC yielded EVs from serum with comparable purity to SEC and similar apolipoprotein B content. Overall, MFC presents an advancement in EV purification yielding EVs with high recovery, purity, and functionality, and offers an accessible improvement to researchers currently employing SEC. [ABSTRACT FROM AUTHOR]

Polymerized impurities in β -lactam antibiotics can induce allergic reactions, which seriously threaten the health of patients. In order to study the polymerized impurities in cefoxitin sodium for injection, a novel approach based on the use of two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry (2D-LC-TOF MS) was applied. In the 1st dimension, high performance size exclusion chromatography (HPSEC) with a TSK-G2000SWxl column was employed. Column switching was applied for the desalination of the mobile phase used to separate polymerized impurities in the 1st dimension before they were transferred to the 2nd dimension which utilized reversed phase liquid chromatography (RP-LC) and TOF MS for further structural characterization. The structures of four polymerized impurities (which were all previously unknown) in cefoxitin sodium for injection were deduced based on the MS2 data. One novel polymerized impurity (PI–I), with 2H less than the molecular weight of two molecules of cefoxitin (M r. 852.09), was found to be the most abundant (>50 %) in almost all the samples examined and could be regarded as the marker polymer of cefoxitin sodium for injection. This work also showed the great potential of the 2D-LC-TOF MS approach in structural characterization of unknown impurities separated with a mobile phase containing non-volatile phosphate in the 1st dimension. [Display omitted] • Novel approach to characterize unstable impurities separated by a non-volatile mobile phase. • Four novel PIs found in cefoxitin based on MSn data. [ABSTRACT FROM AUTHOR]

• SEC-MALS was firstly applied to measure the MW of ULC and SC. • The relationship between relative contents of ULC and PHR were investigated. • The relationship between sizes of ULC and PHR were investigated. • The relationship between heparin MW and the ULC size were investigated. Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy. Antibodies binding to complexes of platelet factor 4 (PF4) and heparin is the trigger of HIT. A method using size exclusion chromatography with multi-angle laser light scattering detector (SEC-MALS) was developed in this work. The soluble ultra-large complex (ULC) was separated from the small complex (SC) and their molecular weights (MWs) were firstly measured. The complexes of PF4 and three heparins with different MW, including unfractionated heparin (UFH), dalteparin (Daltep) and enoxaparin (Eno) were characterized using this method. The contents and the sizes of ULC increased gradually when heparins were added to PF4 to certain amounts. While, they reduced after more heparins were added. It is the first time to measure the MWs of the biggest ULC of PF4-heparins as millions of Dalton. at the proper ratios of PF4 to heparin (PHR). Meanwhile, those mixtures at those certain PHRs induced the higher expression of CD83 and CD14 markers on dendritic cells (DCs) suggesting that they had stronger immunogenicity and is critical for HIT. [ABSTRACT FROM AUTHOR]

The accurate characterization of post-consumer recycled (PCR) polyethylene (PE) and polypropylene (PP) blends is essential for repurposing them into higher-demand applications. This study improves the accuracy of the Temperature Rising Elution Fractionation coupled with Gel Permeation Chromatography (TREFxGPC) approach for both virgin and post-consumer recycled PP/PE blends. Model blends and virgin polymers were used to estimate polymer-specific IR-detector response factors. Moreover, a correction for the molecular weight-dependent amount of PP eluting in the TREF temperature range characteristic of PE was found. Accounting for these factors improved the results of the method, reducing the error in composition determination from 8 % to 2 %. Therefore, our approach provides better quantitative assessment capabilities for recycled blends. Furthermore, the refined TREFxGPC not only reliably determines the PCR PP/PE blend composition but also reveals their bivariate molecular weight and chemical composition distribution (MWD-CCD). Understanding the full microstructure of the blend can lead to superior recycled products, enabling the use of PCR in more demanding applications. • Assessing TREFxGPC's accuracy for PE & PP composition in blends & recyclates. • Adjusting for PP eluting in PE range and polymer-specific detector response. • Method validation on model and recycled blends matches true composition. [ABSTRACT FROM AUTHOR]

Purpose: This study focuses on the effect of length and structure of attached polyethylene glycol (PEG) chain on hydrodynamic radius (Rh) and chromatographic retention of PEGylated protein. To this aim human serum albumin (HSA) as a standard protein was PEGylated site specifically with mPEG-maleimide. Methods: Separated PEG_HSA fractions were analyzed by size exclusion and anion exchange chromatography (AExC). The purity of fractions and the relative mobility of PEGylated and native proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Hydrodynamic radius was determined based on the retention time of fractions on size exclusion chromatography (SEC), and also according to the previously reported equations. Results: A linear relation was shown between the molecular weight of attached PEG and Rh of PEGylated HSA. No significant difference between Rh of proteins modified with linear and branched PEG was shown. In SDS-PAGE, the delaying effect of branched PEG on movement of PEGylated protein was higher than that of linear PEG. Conclusion: As PEGylated HSA and dimer HSA have almost the same size and in SEC they elute at very close retention times, so in this case ion exchange chromatography (IExC) is more effective than SEC in separation of PEGylated HSA. Branched PEG- HSA showed earlier elution on anion exchange chromatography compared to linear PEG-HSA, that this can explain the different shielding effect of various structures of attached PEGs. The smaller size of PEGylated HSA in compare to the sum of the hydrodynamic radiuses of native HSA and attached PEG could be as a result of shielded attachment of polymer around protein. [ABSTRACT FROM AUTHOR]

Aflatoxins (AFs) are one of the most harmful carcinogenic natural toxins that affect food. Crops containing reasonably high oil content may be affected by Aspergillus species and consequently by AF contamination. In this study, a proposed testing method for AF detection in oilseed was developed, validated, and used for a market survey to assess the probabilistic risk exposure caused by consuming contaminated oilseeds including corn, sunflower seed, and soybean. The test method was optimized for selective extraction and then validated for fitness of purpose; the limits of quantification (LOQs) were 0.2, 0.4, 0.2, and 0.2 μg kg−1 for aflatoxin G1 (AFG1), aflatoxin B1 (AFB1), aflatoxin G2 (AFG2), and aflatoxin B2 (AFB2), respectively. The method was linear from the LOQs up to 20 μg kg−1, and its budget of measurement uncertainties were estimated at 25, 24, 26, and 30 for AFG1, AFB1, AFG2, and AFB2, respectively. The contamination levels were from 10,000 body weight (BW) day−1, respectively. The main finding of the present study highlights the possibility of some risk of AF exposure from corn consumption, which may represent a health concern. • Size exclusion chromatography has an excellent and selective extraction of aflatoxins from heavy matrix oilseeds. • According to the consumption rate of corn, the estimated margin of exposure to aflatoxin B1 was 721 body weight (BW) day−1. • There is a possibility of some risk of aflatoxins exposure from corn consumption, which may represent a health concern. [ABSTRACT FROM AUTHOR]

Background: Extracellular vesicles (EVs), known as cell-derived membranous structures harboring a variety of biomolecules, have been widely used in liquid biopsy. Due to the complex biological composition of plasma, plasma RNA omics analysis (RNomics) is easily affected, thus it is necessary to select an optimal strategy from exiting methods according to the performance for intended application.Methods: In this study, four different strategies for EVs isolation were performed and compared (i.e. ultracentrifugation (UC), size exclusion chromatography (SEC), and two most frequently-used commercially available isolation kit (ExoQuick and exoEasy). We compared the yield, purity, PCR quantification of RNAs, miRNA-seq analyses and mRNA-seq analyses of RNAs from EVs isolated using four methods.Results: The results showed that the lowest miRNA binding protein AGO2 (Argonaute-2) and the highest EVs-specific miRNA and lncRNA were observed in EVs obtained through SEC, meanwhile the content of the non-specific miRNA was the lowest. Further RNA-Seq data revealed that RNAs obtained via SEC presented more useful reads for both miRNA and mRNA. Furthermore, the mRNA delivered via SEC tended to have a concentration comparable to the ideal FPKM (Fragments Per Kilobase Million) value.Conclusions: SEC shall be used as an optimal strategy for the isolation of EVs in plasma RNomics analysis. [ABSTRACT FROM AUTHOR]

• Explain the separation mechanism of ultrasound-assisted chromatography (UAC). • Comparatively analyzed ultrasound effects on C18 PEEK and SS columns. • Distinguished the differences between acoustic and temperature effects in UAC. • Ultrasound can be utilized as an external factor in chromatography. Based on the previous ultrasound-assisted chromatography (UAC) studies on plastic and stainless steel (SS) columns, this study explores the UAC mechanism by comparatively analyzing ultrasound effects on plastic and SS columns with C18 stationary phase when separating a mixture of polycyclic aromatic hydrocarbons (PAHs) under various ultrasound intensities. The results showed a substantial difference in H values between the PEEK and SS columns under the influence of ultrasound agitation. Specifically, for the pyrene peak, as the ultrasonic intensity increased from 0% to 100% of 900 W, the H values of the SS column slightly rose from 8.82 μm to 9.86 μm. Conversely, the corresponding values for the PEEK column exhibited a significant 12-fold increase from 11.5 μm to 134 μm. The findings demonstrated poor penetration of ultrasound energy through the SS column, and the temperature rise of the medium induced by the ultrasound was the primary contributing factor to PAH separation. However, ultrasound easily penetrated through the plastic column, resulting in acoustic cavitation within the C18 polyether ether ketone (PEEK) column. Cavitation induced heat generation and contributed to a decrease in retention time and the magnitude of peak broadening or distortion, depending on the specific ultrasonic energy. Based on the estimated change in inlet temperature of the PEEK column due to an acoustic effect, the comparison with temperature effects under non-sonic conditions consistently demonstrated a stronger acoustic effect in reducing the retention time, by 2–9%, depending on specific peaks and pairs. We revisited the previously described separation mechanism of ultrasound-assisted ion chromatography and conjoined with our findings to infer and establish a thorough explanation for the previously unexplained separation mechanism of chiral separation and size exclusion chromatography by UAC using SS columns. [ABSTRACT FROM AUTHOR]

The analysis of complex mixtures of closely related species is quickly becoming a bottleneck in the development of new drug substances, reflecting the ever-increasing complexity of both fundamental biology and the therapeutics used to treat disease. Two-dimensional liquid chromatography (2D-LC) is emerging as a powerful tool to achieve substantial improvements in peak capacity and selectivity. However, 2D-LC suffers from several limitations, including the lack of automated multicolumn setups capable of combining multiple columns in both dimensions. Herein, we report an investigation into the development and implementation of a customized online comprehensive multicolumn 2D-LC-DAD-MS setup for screening and method development purposes, as well as analysis of multicomponent biopharmaceutical mixtures. In this study, excellent chromatographic performance in terms of selectivity, peak shape, and reproducibility were achieved by combining reversed-phase (RP), strong cation exchange (SCX), strong anion exchange (SAX), and size exclusion chromatography (SEC) using sub-2-μm columns in the first dimension in conjunction with several 3.0 mm × 50 mm RP columns packed with sub-3-μm fully porous particles in the second dimension. Multiple combinations of separation modes coupled to UV and MS detection are applied to the LC × LC analysis of a protein standard mixture, intended to be representative of protein drug substances. The results reported in this study demonstrate that our automated online multicolumn 2D-LC-DAD-MS workflow can be a powerful tool for comprehensive chromatographic column screening that enables the semi-automated development of 2D-LC methods, offering the ability to streamline full visualization of sample composition for an unknown complex mixture while maximizing chromatographic orthogonality. [ABSTRACT FROM AUTHOR]

A complex mixture of peptides plays a key role in the regulation of the immune system; different sources as raw materials mainly from animals and vegetables have been reported to provide these extracts. The batch-to-batch product consistency depends on in-process controls established. However, when an immunomodulator is a customized product obtained from the same volunteer who will receive the product to personalize the treatment, the criteria to establish the consistency between volunteers are different. In this sense, it is expected to have the same molecular weight range although the profile of peptide abundance is different. Here, we characterized the peptide profile of three extracts of an immunomodulator obtained from the urine of different volunteers suffering from three different diseases (i.e., allergic rhinitis, rheumatoid arthritis, and chronic rhinopharyngitis), using size exclusion chromatography (SEC) and mass spectrometry (MS). The peptides contained in the immunomodulators were stable after six months, stored in a refrigerator. Our results showed a chromatographic profile with the same range of low molecular weight (less than 17 kDa) in all analyzed samples by SEC; these results were also confirmed by MS showing an exact mass spectrum from 3 to 13 kDa. The fact that the peptide profiles were conserved during a six-month period at refrigeration conditions (2 to 8°C) maintaining the quality and stability of the immunomodulator supports the notion that it might be an alternative in the treatment of chronic hypersensibility disorders. [ABSTRACT FROM AUTHOR]

Exosomes are small nanovesicles released by the cells with a potential therapeutic application. A semi-continuous size exclusion chromatography process was developed to support the purification of these nanovesicles that can be used as drug delivery vehicles, diagnostic tools and vaccines. • Semi-continuous process using size exclusion chromatography for extracellular vesicles (EV) purification. • The recovery yield was increased from 42% (batch) to 83% in the developed process. • Specific productivity was increased by 167% and buffer consumption was reduced by a 2.7-fold. • EV cup-shaped morphology and size were maintained during the purification process. Extracellular vesicles, more particularly exosomes, are nanostructures of great medical interest. Similarly to other complex biopharmaceuticals such as virus, the purification of these nanovesicles is still a considerable challenge. Here we describe a proof-of-concept purification strategy based on the well-established size exclusion chromatography technique, operated in a semi-continuous mode. A two-column system was designed and experimentally validated. Immunoblotting for protein exosome markers and electron microscopy indicated the presence of exosomes, with its characteristic cup-shaped morphology. The isolated nanovesicles have a mode size of 128 nm and a concentration and size distribution very similar between the collected products. Importantly, after comparison with batch chromatography, we were able to increase the yield up to 83%, specific productivities in 167% and a 2.7-fold reduction in buffer consumption with the semi-continuous operation. Finally, we demonstrate that the developed process can be potentially applied in the large-scale production of exosome-based therapeutics. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) include a variety of nanosized vesicles released to the extracellular microenvironment by the vast majority of cells transferring bioactive lipids, proteins, mRNA, miRNA or non-coding RNA, as means of intercellular communication. Remarkably, among other fields of research, their use has become promising for immunomodulation, tissue repair and as source for novel disease-specific molecular signatures or biomarkers. However, a major challenge is to define accurate, reliable and easily implemented techniques for EV isolation due to their nanoscale size and high heterogeneity. In this context, differential ultracentrifugation (dUC) has been the most widely used laboratory methodology, but alternative procedures have emerged to allow purer EV preparations with easy implementation. Here, we present and discuss the most used of the different EV isolation methods, focusing on the increasing impact of size exclusion chromatography (SEC) on the resulting EV preparations from in vitro cultured cells-conditioned medium and biological fluids. Comparatively, low protein content and cryo-electron microscopy analysis show that SEC removes most of the overabundant soluble plasma proteins, which are not discarded using dUC or precipitating agents, while being more user friendly and less time-consuming than gradient-based EV isolation. Also, SEC highly maintains the major EVs' characteristics, including vesicular structure and content, which guarantee forthcoming applications. In sum, together with scaling-up possibilities to increase EV recovery and manufacturing following high-quality standards, SEC could be easily adapted to most laboratories to assist EV-associated biomarker discovery and to deliver innovative cell-free immunomodulatory and pro-regenerative therapies. [ABSTRACT FROM AUTHOR]

Size-exclusion chromatography (SEC) with wide application in peptide analysis presents challenges in determination of molecular weight distribution due to the relatively low resolution. In this study, a stop-flow reversed phase liquid chromatography (RPLC) × SEC system was constructed, aiming at improving the peptide separation in SEC. As the chromatographic dispersion during stop-flow operation might contribute to the band broadening in the first dimension (1st D) RPLC, the effects of different stop-flow operational parameters on the additional band broadening were quantitatively evaluated. Unlike analytes of large molecular size or long retention time with low effective diffusion coefficient ( D eff ), additional band broadening was evidently observed for analytes of small molecular size and short retention time (high D eff ). Therefore, optimal flow rate, low column temperature and short stop-flow time were suggested for analyzing small molecules of short retention time. The established stop-flow two-dimensional liquid chromatography (2D-LC) was further tested on protein hydrolysates. The resolution was evidently improved for both heart-cutting and comprehensive 2D-LC analysis (despite additional band broadening in RPLC). Compared with heart-cutting analysis with higher 1st D resolution for selective fractions, comprehensive analysis could provide more complete information about the molecular weight distribution of the eluting solutes along RPLC. [ABSTRACT FROM AUTHOR]

• Capillary-channeled polymer fibers provide high throughput as 2D in 2DLC. • Fiber columns in 2D eliminates the need for UPLC hardware in the second dimension. • SEC x RP with fiber columns provides for potential implementation for ESI sampling. • PET and PP fibers exhibit different levels of selectivity, under RP conditions. • RP x RP with fiber columns provides orthogonality in chemical information. Polymeric materials are readily available, durable materials that have piqued the interest of many diverse fields, ranging from biomedical engineering to construction. The physiochemical properties of a polymer dictate the behavior and function, where large polydispersity among polymer properties can lead to problems; however, current polymer analysis methods often only report results for one particular property. Two-dimensional liquid chromatography (2DLC) applications have become increasingly popular due to the ability to implement two chromatographic modalities in one platform, meaning the ability to simultaneously address multiple physiochemical aspects of a polymer sample, such as functional group content and molar mass. The work presented employs size exclusion chromatography (SEC) and reversed-phase (RP) chromatography, through two coupling strategies: SEC x RP and RP x RP separations of the water-soluble polymers poly(methacrylic acid) (PMA) and polystyrene sulfonic acid (PSSA). Capillary-channeled polymer (C-CP) fiber (polyester and polypropylene) stationary phases were used for the RP separations. Particularly attractive is the fact that they are easily implemented as the second dimension in 2DLC workflows due to their low backpressure (<1000 psi at ∼70 mm sec−1) and fast separation times. In-line multi-angle light scattering (MALS) was also implemented for molecular weight determinations of the polymer samples, with the molecular weight of PMA ranging from 5 × 104 to 2 × 105 g mol−1, while PSSA ranges from 105 to 108 g mol−1. While the orthogonal pairing of SEC x RP addresses polymer sizing and chemistry, this approach is limited by long separation times (80 min), the need for high solute concentrations (PMA = 1.79 mg mL−1 and PSSA = 0.175 mg mL−1 to yield comparable absorbance responses) due to on-column dilution and subsequently limited resolution in the RP separation space. With RP x RP couplings, separation times were significantly reduced (40 min), with lower sample concentrations (0.595 mg mL−1 of PMA and 0.05 mg mL−1 of PSSA) required. The combined RP strategy provided better overall distinction in the chemical distribution of the polymers, yielding 7 distict species versus 3 for the SEC x RP coupling. [ABSTRACT FROM AUTHOR]

Abstract: Metal–metal cooperation is integral to the function of many enzymes and materials, and model complexes hold enormous potential for providing insights into the capabilities of analogous multimetallic cores. However, the selective synthesis of heterobimetallic complexes still presents a significant challenge, especially for systems that hold the metals in close proximity and feature open or reactive coordination sites for both metals. To address this issue, a rigid, naphthyridine‐based dinucleating ligand featuring distinct binding environments was synthesized. This ligand enables the selective synthesis of a series of MIICuI bimetallic complexes (M=Mn, Fe, Co, Ni, Cu, Zn), in which each metal center exclusively occupies its preferred binding pocket, from simple chloride salts. The precision of this selectivity is evident from cyclic voltammetry, ESI‐MS and anomalous X‐ray diffraction measurements. [ABSTRACT FROM AUTHOR]

Emerging persistent organic pollutants (ePOPs) include polybrominated diphenyl ethers (PBDEs) and perfluorooctane sulfonyl fluoride/perfluorooctane sulfonate (POSF/PFOS), which are newly listed in the Stockholm Convention. Other ePOPs, which have not been regulated, include organophosphate flame retardants (PFRs), novel brominated flame retardants (NBFRs) and other perfluoroalkyl substances (PFASs). Often ePOPs data related to occurrence, toxicity, impact or environmental behavior are insufficient or inadequate because of the lack of proper analytical methods to obtain them. Thus, a critical review of the analytical procedures proposed in the last six years (2011–2017) for determining ePOPs by chromatographic methods in the different compartments of the aquatic ecosystems is presented. The overall analytical procedure, from sampling to final determination, is emphasized presenting recent developments in the extraction, pre-concentration, and instrumental detection needed for the accurate quantification of ePOPs in environmental samples. Finally, this review examines the basic challenges we face in order to anticipate future directions and urgent needs of this field. [ABSTRACT FROM AUTHOR]

Plant polysaccharides have numerous medicinal functions. Due to the differences in their origins, regions of production, and cultivation conditions, the quality and the functions of polysaccharides can vary significantly. They are macromolecules with large molecular weight (MW) and complex structure, and pose great challenge for the analytical technology used. Taking Dendrobium officinale (DO) from various origins and locations as model samples. In this investigation, mechanochemical extraction method was used to successfully extract polysaccharides from DO using water as solvent, the process is simple, fast (40 s) and with high yield. The MWs of the intact saccharides from calibration curve and light scattering measurement were determined and compared after separation with size exclusion chromatography (SEC). The large polysaccharide was acid hydrolyzed to oligosaccharides and the products were efficiently separated and identified using liquid chromatography coupled to a high resolution tandem mass spectrometry (LC–MS 2 ). Obvious differences were observed among LC–MS 2 chromatograms of digested products, and the chemical structures for the products were proposed based on accurate mass values. More importantly, isomeric digested carbohydrate compounds were explored and characterized. All the chromatographic and mass spectrometric results in this study provided a multi-dimensional characterization, fingerprint analysis, and molecular structure level assessment of plant polysaccharides. [ABSTRACT FROM AUTHOR]

Abstract: Chemosensors (CSs) with dynamically tunable detection ranges have important significance for their expansion in practical applications; however, most CSs possess an unchangeable detection limit. In this work, we report the first example of a supramolecular polymer vesicle (SPV) chemosensor with a dynamically tunable detection range. SPVs containing porphyrin (PP) moieties and β‐cyclodextrin (β‐CD)/azobenzene (Azo) host–guest interactions were first constructed. The obtained SPVs were used to detect Zn2+ with a high selectivity and sensitivity over a wide detection limit range of 8.67×10−9 to 1.99×10−11 under UV light irradiation. The corresponding sensing mechanism was attributed to the synergistic effects of the triple noncovalent interactions, including the metal‐ligand coordination of PP/Zn2+ and the double host–guest interactions of β‐CD/Azo and β‐CD/PP. [ABSTRACT FROM AUTHOR]

The article examines the use of an automated process in online peak fractionation and reduction of therapeutic antibodies using subsequent quadrupole time-of-flight mass spectrometry (QTOF-MS) characterization. Aspartic acid or asparagine residues in the light and heavy chain result in succinimide under acidic conditions. Characterization of charge and size variants is needed to produce the product knowledge that forms the basis for critical quality attribute (CQA) assessment.

Abstract: Small‐angle X‐ray scattering (SAXS) is a relatively simple experimental technique that provides information on the global conformation of macromolecules in solution, be they fully structured, partially, or extensively unfolded. Size exclusion chromatography in line with a SAXS measuring cell considerably improves the monodispersity and ideality of solutions, the two main requirements of a “good” SAXS sample. Hydrogen/deuterium exchange monitored by mass spectrometry (HDX‐MS) offers a wealth of information regarding the solvent accessibility at the local (peptide) level. It constitutes a sensitive probe of local flexibility and, more generally, of structural dynamics. The combination of both approaches presented here is very powerful, as illustrated by the case of RD, a calcium‐binding protein that is part of a bacterial virulence factor. [ABSTRACT FROM AUTHOR]

The transcriptional regulator BrlR is a member of the MerR family of multidrug transport activators in Pseudomonas aeruginosa . Recent study indicates that BrlR is a novel 3′,5′-cyclic diguanylic acid (c-di-GMP) receptor and can be activated by c-di-GMP. To gain insight into BrlR function, we determined the structure of BrlR with c-di-GMP complex structure to 2.5 Å. The structure and size exclusion chromatography (SEC) data revealed BrlR forms a tetramer and each BrlR protomer consists of three parts, DNA-binding domain, a coiled-coil region and GyrI-like domain. There are two different c-di-GMP binding sites located mainly at the DNA binding domain of each BrlR protomer and do not overlap with the GyrI-like domain. The drug-binding pocket in GyrI-like domain is much conserved indicating it can also bind flat-shaped molecules like other multidrug resistance (MDR) proteins. [ABSTRACT FROM AUTHOR]

• Simulated moving bed chromatography can be performed at a microliter scale. • Custom-made 3D printed valve reduces overall dead volume to 358 µL. • Pressure-driven closed-loop flow control is a suitable flow source. • Desalting level of up to 99% for continuous protein desalting • Feed flow rate as low as 15 µL/min. In the 1960s, chromatography processes were revolutionized by the invention of simulated moving bed chromatography. This method not only enhances the separation performance and resin utilization in comparison to batch-chromatography, it has also a much lower buffer consumption. While simulated moving bed chromatography nowadays is applied for a wide range of industrial applications, it was never transferred to the micro-scale (in regards to column and system volume). In our opinion a micro simulated moving bed chromatography system (µSMB) would be a useful tool for many applications, ranging from early process development and long term studies to downstream processing of speciality products. We implemented such a µSMB with a 3D printed central rotary valve and a microfluidic flow controller as flow source. We tested the system with a four zone open loop setup for the separation of bovine serum albumin and ammonium sulfate with size exclusion chromatography. We used four process points and could achieve desalting levels of BSA ranging from 94% to 99%, with yields ranging form 65% to 88%. Thus, we were able to achieve comparable results to common lab scale processes. With a total dead volume of 358 µL, including all sensors, connections and the valve, this is, to the best of our knowledge, the smallest SMB system that was ever built and we were able to perform experiments with feed flow rates reaching as low as 15 µL/min. [ABSTRACT FROM AUTHOR]

Serine/threonine-protein kinase-like protein ARABIDOPSIS CRINKLY4 (ACR4), a transmembrane protein of Arabidopsis thaliana , plays important roles in cell division and differentiation. Although accumulating studies shed light on the function of ACR4, the structure and catalytic mechanism of ACR4 remain to be elucidated. Here, we report the purification and enzymatic properties of the intracellular kinase domain (residues 464–799) of ACR4 (ACR4 IKD ). Through Ni-affinity chromatography and gel filter chromatography methods, we successfully obtain high-purity ACR4 IKD protein from Escherichia coli . Dynamic light scattering and gel-filtration methods reveal that ACR4 IKD distributes with high homogeneity and exists as a monomer in solution. In addition, the ACR4 IKD protein has typical kinase activity with myelin basic protein (MBP) as the substrate. Our study may lay the foundation for structure determination of ACR4 IKD and further functional research, for example, screening significant substrates of ACR4 in Arabidopsis thaliana . [ABSTRACT FROM AUTHOR]

A method for assaying sodium caprylate content using gel filtration HPLC and recording of the refractometric detector signal is described. Validation studies demonstrated the suitability of the method. Inclusion of a spectrophotometer in the chromatography system provided for monitoring two parameters ('Sodium caprylate' and 'Polymers and aggregates') in a single test. [ABSTRACT FROM AUTHOR]

A heterogalactan, named ACW0, was extracted from Antrodia camphorata and purified by anion exchange and gel permeation chromatography. It was composed of galactose (94.98%), traces of mannose (2.41%), and fucose (2.61%), with its molecular weight estimated to be 13.5 k Da. The polysaccharide ACW0 was shown to be a mannofucogalactan with a backbone chain of α-D-1,6-linked Gal, attached by a non-reducing terminal α-D-Man and α-L-Fuc on C-2 of nearly every six α-D-1,6-linked Gal residues. A sulfated polysaccharide, ACW0-Sul was achieved by the chlorosulfonic acid-pyridine method. Compared with the native polysaccharide, ACW0-Sul could disrupt tube formation and migration as well as cell growth of human microvascular endothelial cells (HMEC-1) dose-dependently. Further studies revealed that phosphorylation of Extracellular Regulated Protein Kinases (Erk) and Focal Adhesion Kinase (FAK) were significantly inhibited by ACW0-Sul. These results suggested that ACW0-Sul could be a potent candidate for anti-angiogenic agent development. [ABSTRACT FROM AUTHOR]

Detection of higher-order aggregates (HOA) using size-exclusion chromatography (SEC) was found to be variable for a basic protein, using exposed-silanol or diol-silica-based SEC columns. Preparations of the tetrameric biopharmaceutical enzyme Erwinia chrysanthemi l -asparaginase (ErA), which has an isoelectric point of 8.6, were analysed using a diol-silica SEC column. Although the proportions of ErA main peak and octamer species were unaffected, HOA recovery and detection were extremely variable and had poor agreement with an orthogonal measurement technique, analytical ultracentrifugation (AUC). The observation that only HOA was selectively affected by non-specific silanol interactions was unexpected, so alternatives were sought. Coated-silica SEC columns improved the resolution and reproducibility of HOA detection for this alkaline-pI protein, and improved the agreement of HOA with the AUC method. Basic proteins, such as ErA, should be thoroughly evaluated in SEC method development, to ensure that resolution of larger aggregate species is not compromised. [ABSTRACT FROM AUTHOR]

α-Amylase from Cryptococcus flavus was previously purified after exchange chromatography on Q-Sepharose and gel filtration on Sephacryl S-100. The enzyme was purified 128-fold with a recovery rate of 81% of activity. The molecular mass of the α-amylase was 67 kDa as determined by SDS-PAGE. The enzyme was characterized after immobilization on glass tubes. The immobilization yield, efficiency, and activity recovery of α-amylase were 79, 57, and 45%, respectively. The optimum pH of the immobilized enzyme was shifted to a more acidic pH (4.5) compared with the free enzyme (5.5). The optimum temperature of the immobilized enzyme was the same for the free enzyme (50°C), but it showed a higher thermal stability. The immobilized enzyme showed activity until the 10th cycle, maintaining 47% of initial activity, and was capable of hydrolyzing starch from potato, wheat, corn, and cassava. The main products released from starch were maltose, maltotriose, and maltotetraose. [ABSTRACT FROM AUTHOR]

A comparison is made using size-exclusion chromatography (SEC) of synthetic polymers between fully porous particles (FPPs) and superficially porous particles (SPPs) with similar particle diameters, pore sizes and equal flow rates. Polystyrene molecular weight standards with a mobile phase of tetrahydrofuran are utilized for all measurements conducted with standard HPLC equipment. Although it is traditionally thought that larger pore volume is thermodynamically advantageous in SEC for better separations, SPPs have kinetic advantages and these will be shown to compensate for the loss in pore volume compared to FPPs. The comparison metrics include the elution range (smaller with SPPs), the plate count (larger for SPPs), the rate production of theoretical plates (larger for SPPs) and the specific resolution (larger with FPPs). Advantages to using SPPs for SEC are discussed such that similar separations can be conducted faster using SPPs. SEC using SPPs offers similar peak capacities to that using FPPs but with faster operation. This also suggests that SEC conducted in the second dimension of a two-dimensional liquid chromatograph may benefit with reduced run time and with equivalently reduced peak width making SPPs advantageous for sampling the first dimension by the second dimension separator. Additional advantages are discussed for biomolecules along with a discussion of optimization criteria for size-based separations. [ABSTRACT FROM AUTHOR]

In this study, to investigate the evolution of dissolved organic matter (DOM) in starch processing wastewater (SPW) during the treatment of a sedimentation and biological hybrid system, the DOM was sampled at different treatment stage, and characterized by gel permeation chromatography (GPC), fluorescence excitation-emission matrix (EEM) and UV–Vis. The chromatograms demonstrated that the majority of molecules in the influent had a molecular weight (MW) of less than 0.5 kDa, while that of the oxic basin was mainly in the range of 0.5–2 kDa. The MW distribution became narrow along the treatment line. The proteinaceous matters with simple structure were more easily biodegraded during the treatment process. Most protein-like material could be effectively decomposed by the activities of anaerobic and aerobic bacteria. The microbial byproduct-like substances and humic acid-like materials were the main components of DOM in the effluent. The parameters obtained from the analysis were clustered into two groups, which represent the different treatment state of SPW. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Schisandra chinensis (Turcz.) Baill. ( S. chinensis ), locally known as “Wuweizi”, has been used in the treatment of chronic cough as prescription medications of Traditional Chinese Medicine for thousands of years. However, the components of antitussive activity of S. chinensis and the mechanism are poorly understood. Aim of the study This study aims to investigate the antitussive activity of polysaccharides extracted from S. chinensis . Materials and methods S. chinensis fruit polysaccharide-1 (SCFP-1) was extracted by 95% ethanol and distilled water successively, and then the water extraction was isolated with chromatographic columns. The preliminary characterization of SCFP-1 was analyzed by gel permeation chromatography (GPC), gas chromatography–mass spectrometry (GC–MS) and some other recognized chemical methods. Antitussive potential of SCFP-1 was estimated at dose of 250, 500, and 1000 mg/kg respectively by peroral administration in a guinea pigs model with cough hypersensitivity induced by cigarette smoke (Chronic cough model) or acute cough guinea model induced by citric acid (Acute cough model). Also, the time-dependent antitussive effect of SCFP-1 were evaluated with acute cough model, and compared with codeine. Results The molecular of SCFP-1 was 3.18×10 4 Da, mainly being composed of glucose and arabinose (66.5% and 29.4%, respectively). Peroral administration of SCFP-1 at 250, 500, and 1000 mg/kg showed remarkable suppressive effects respectively on cough in both of chronic cough model and acute cough model. Meanwhile, inflammatory cell in BALF and some typical characteristics of nonspecific airway inflammation in animals exposed to CS was significantly attenuated after pretreatment with SCFP-1. The cough suppression of SCFP-1 (500 mg/kg) stablly lasted during the whole 5 h of time-dependent experiment, while no positive effect was observed after 300 min of oral administration of codeine. Conclusions SCFP-1 is one of the antitussive components of S. chinensis . [ABSTRACT FROM AUTHOR]

Hyperbranched polyethylenimine (HPEI) was modified with glycidol, resulting in hydroxyl terminated HPEI (HPEI-OH). Subsequently, HPEI-OH was used as the macroinitiator to initiate the anionic ring-opening polymerization of glycidol, realizing the hyperbranched grafting copolymer with HPEI as core and hyperbranched polyglycerol (HPG) as shell (HPEI- g -HPG) through the 'grafting from' method. The diagrams from gel permeation chromatography (GPC) showed that the products were a mixture of copolymers and homopolymers. A two-step purification method was developed to separate HPEI- g -HPGs from the mixture, which was much more effective than the normally-adopted dialysis method. The hyperbranched grafting polymerization condition was optimized and the maximal grafting efficiency was found to be ca. 30%. Under the optimal grafting polymerization condition, we prepared a series of HPEI- g -HPG copolymers that have the same HPEI core, but have a different number of glycidol units in the HPG shell. The detailed structural information of these copolymers was deduced from 1 H NMR, inverse gated decoupled 13 C NMR and GPC measurements. Although no traditional fluorophores existed in HPEI- g -HPGs, HPEI- g -HPGs could emit blue fluorescence centered at ca. 470 nm. The fluorescence intensity was influenced pronouncedly by the thickness of HPG shell, pH, oxidation time, the amount of THF in the solvent mixture. The detailed characterizations and analyses supported that the luminogen of HPEI- g -HPGs was tertiary amine oxide. [ABSTRACT FROM AUTHOR]

Two graft copolymer samples of identical average composition were synthesized by grafting living polystyrene anions onto a broadly distributed PMMA backbone. Size exclusion chromatography (SEC) with only RI-detection, SEC with viscometry and light scattering detection, SEC with UV and RI dual detection, gradient chromatography and 2-dimensional chromatography were applied to compare the information that can be obtained by the different techniques. While only limited information was retrieved by conventional SEC or SEC with molar mass sensitive detection, SEC with UV and RI revealed different chemical heterogeneity of the samples. Using gradient chromatography and 2-dimensional chromatography it was possible to identify non-grafted side chains and unreacted parent PMMA besides the actual graft copolymer molecules. While in one sample a heavily grafted product was formed besides non-grafted PMMA, the second sample did not contain ungrafted PMMA but a graft product of lower grafting density. The different product distributions were explained by the different synthetic procedures. [ABSTRACT FROM AUTHOR]

The depolymerisation of porcine mucosal heparan sulfate under the action of heparin lyases and analysis by size-exclusion chromatography (SEC) is described. Heparan sulfate treated to enzymic bond scission producing a Δ 4,5 double-bond and quantified by SEC with ultraviolet-visible (UV) spectroscopic detection (230 nm) indicated that the majority of the biopolymer (>85%) was reduced to disaccharides (degree of polymerisation (DP) = 2). However, analysis of the SEC eluant using refractive index (RI), which reflects the mass contribution of the oligosaccharides rather than the molar response of a UV chromophore, indicated that a considerable proportion of the digested HS, up to 43%, was present with DP >2. This was supported by a mass balance analysis. These results contradict the accepted literature where “complete digestion” is routinely reported. Herein we report on the composition and methodology utilised to ascertain the extent of depolymerization and disaccharide composition of this important biopolymer. [ABSTRACT FROM AUTHOR]

• SEC packing materials with three different bondings were prepared. • The three materials were characterized and compared. • Non-specific interactions were systematically studied. • The new HO-PEO phase was clearly the most favourable one. • A fast platform method (3 min analysis time) has been developed for protein SEC. Size exclusion chromatography has become an essential tool for the protein therapeutics industry. Conceptually, it is a simple form of chromatography that is driven by entropy and sieving effects. An ideal size exclusion column would exhibit no adsorptive interactions between its internal surfaces and the solutes being analysed, but that is not easily achieved. To this end, we have studied the utility of three unique packing materials in pursuit of additional column chemistries that might be less prone to interacting with proteins. These packing materials were each prepared from bridged ethylene hybrid organic/inorganic particles but uniquely derivatized into either hydroxy terminated PEO bonded, methoxy terminated PEO bonded, or diol bonded packing materials. All three materials were packed into column hardware modified with hydrophilic hybrid surface technology (h-HST) so that packing material effects could be more clearly observed without any influence from the secondary interactions that can originate from metal hardware. Non-specific interactions were compared for various challenging protein samples in the presence of ammonium acetate (volatile) and phosphate buffered saline (non-volatile) buffers. It was reconfirmed that the h-HST column hardware mitigates a majority of non-desired secondary interactions. However, during studies on hydrophobic interactions, the new hydroxy terminated PEO packing material showed clear benefit to obtaining higher apparent recoveries to better ensure accurate aggregate quantitation. Further experiments were explored to show that a hydroxy terminated PEO column could be effectively paired with a mobile phase comprised of standard strength phosphate buffered saline to make a fast platform method capable of baseline resolving monoclonal antibody monomer and aggregate peaks within a 3 min analysis time. [ABSTRACT FROM AUTHOR]

Surface plasmon resonance (SPR) is an optical technique that measures biomolecular interactions. Stand-alone SPR cannot distinguish different binding components present in one sample. Moreover, sample matrix components may show non-specific binding to the sensor surface, leading to detection interferences. This study describes the development of coupled size-exclusion chromatography (SEC) SPR sensing for the separation of sample components prior to their on-line bio-interaction analysis. A heterogeneous polyclonal human serum albumin antibody (anti-HSA) sample, which was characterized by proteomics analysis, was used as test sample. The proposed SEC-SPR coupling was optimized by studying system parameters, such as injection volume, flow rate and sample concentration, using immobilized HSA on the sensor chip. Automated switch valves were used for on-line regeneration of the SPR sensor chip in between injections and for potential chromatographic heart cutting experiments, allowing SPR detection of individual components. The performance of the SEC-SPR system was evaluated by the analysis of papain-digested anti-HSA sampled at different incubation time points. The new on-line SEC-SPR methodology allows specific label-free analysis of real-time interactions of eluting antibody sample constituents towards their antigenic target. [ABSTRACT FROM AUTHOR]

Band broadening in size-exclusion chromatography (SEC) is always present to some extent. Broadening effects on averages such as the weight- and number average molecular weights ( M W ¯ and M n ¯ respectively) are minimal with modern SEC systems. However, broadening distorts the shape of the true molecular weight distribution (MWD), which causes problems if one wants to compare the detailed form of the MWD to a model. An addition to current methods for overcoming this problem is presented. One starts with a sufficiently wide range of samples whose exact values of M n ¯ and M W ¯ have been measured by non-SEC methods (e.g. by fluorimetry and light scattering, respectively, of the sample without size separation). A true (unbroadened) molecular weight distribution for a sample can be obtained by deconvolution (here using a maximum-entropy algorithm) by fitting SEC data for these samples to these exact M n ¯ and M W ¯ values to find the values of the parameters in a sufficiently flexible assumed broadening function. This was modelled using simulated band broadening and subsequent deconvolution, with the broadening parameters least-squares fitted to the “exact” sets of values of M n ¯ and M W ¯ . The results show that if these M n ¯ and M W ¯ values are for a series of broad (not narrow) standards covering a sufficient range of molecular weight, then after deconvolution, a good representation of the original molecular weight distribution used in the simulation is obtained. The method should prove useful for water-soluble polymers, for which it is often difficult to obtain narrow standards of a wide range of molecular weight, as required in a number of well-established methods for correcting for band broadening. [ABSTRACT FROM AUTHOR]

A purification scheme based on hydrophobic interaction chromatography was developed to separate inactivated foot-and-mouth disease virus (FMDV) from crude supernatant. About 92% recovery and 8.8-fold purification were achieved on Butyl Sepharose 4FF. Further purification on Superdex 200 resulted in another 29-fold purification, with 92% recovery. The columns were coupled through an intermediate ultrafiltration unit to concentrate the virus. The entire process was completed in about 3.5 h, with 75% final FMDV recovery, and 247-fold purification. The final product had purity above 98%, with over 99.5% of host cell DNA removed. High-performance size exclusion chromatography (HPSEC), Western blot, dynamic light scattering (DLS), and transmission electron microscopy (TEM) indicated that the purified virus contained the required antigen, and was structurally intact with a spherical shape and a particle size of 28 nm. [ABSTRACT FROM AUTHOR]

Average molecular weights and polydispersity indexes are some of the most important parameters considered in the polymer characterization. Usually, gel permeation chromatography (GPC) and multi angle light scattering (MALS) are used for this determination, but GPC values are overestimated due to the dispersion introduced by the column separation. Several procedures were proposed to correct such effect usually involving more complex calibration processes. In this work, a new method of calculation has been considered including diffusion effects. An equation for the concentration profile due to diffusion effects along the GPC column was considered to be a Fickian function and polystyrene narrow standards were used to determine effective diffusion coefficients. The molecular weight distribution function of mono and poly disperse polymers was interpreted as a sum of several Fickian functions representing a sample formed by only few kind of polymer chains with specific molecular weight and diffusion coefficient. Proposed model accurately fit the concentration profile along the whole elution time range as checked by the computed standard deviation. Molecular weights obtained by this new method are similar to those obtained by MALS or traditional GPC while polydispersity index values are intermediate between those obtained by the traditional GPC combined to Universal Calibration method and the MALS method. Values for Pearson and Lin coefficients shows improvement in the correlation of polydispersity index values determined by GPC and MALS methods when diffusion coefficients and new methods are used. [ABSTRACT FROM AUTHOR]

Herein, a striking anion-tunnel transfer effect was demonstrated in 1-butyl-3-methylimidazolium benzoate (BmimPhCOO) ionic liquid, so a rapid, mild and efficient benzoylation of cellulose is accomplished under catalyst-free condition in BmimPhCOO. In a wide temperature range of 20–80 °C, the equilibrium of reaction is reached within only 15 min, which is much faster than the reported acylation of cellulose. Furthermore, the resultant cellulose tribenzoates have excellent solubility in conventional organic solvents, thus they can be used to accurately reflect the molecular weight and dispersity index of cellulose raw materials by gel permeation chromatography. This method is suitable for various cellulose. Therefore, we found a new principle to realize the extremely-rapid acylation of cellulose, and proposed an effective approach to accurately determine the molecular weight parameters of cellulose. A new principle to realize the rapid acylation of cellulose was found to establish an effective strategy for accurately characterizing the molecular weight parameters of cellulose. [Display omitted] [ABSTRACT FROM AUTHOR]

• 2D SEC-CEX-MS allows multiplex analysis of mAb aggregates and charge variants in a single method. • 2D SEC-CEX-MS streamlines product development in industry. • 2D SEC-CEX-MS is applied to understand mAb forced degradation pathways. Monoclonal antibodies (mAbs) are extremely complex due to the presence of structural modifications resulting from enzymatic and chemical reactions such as glycosylation, glycation, deamidation, isomerisation, oxidation, aggregation and fragmentation. Size and charge variants analysis are carried out from the early stages of drug development throughout product lifetime to investigate product degradation pathways and optimise process conditions. However, conventional analytical workstreams for size and charge variant characterization are both time and sample demanding, requiring the application of multiple analytical methods. This study presents the development of a novel 2D-LC/MS approach combining both aggregate and charge variant profiling of a mAb candidate in a single method. Aggregate quantification was performed in the first dimension (1D) by size exclusion chromatography SEC, followed by online fraction transfer of the monomer peak to the second dimension (2D) by a heart-cutting for charge variant analysis by cation exchange chromatography (CEX). Aiming to maximise the information obtained from minimal sample and time required for analysis, a salt-based separation with UV detection was developed for supporting the processing of a large number of samples to facilitate high-throughput process development (HTPD). In addition, a mass spectrometry (MS) compatible SEC-CEX separation was developed enabling online charge variant peak identification. This study presented the ability to multiplex mAb size and charge variants analysis by coupling SEC with CEX in a 2D-LC set-up. To date, this is the first 2D SEC-CEX-UV(-MS) application for intact mAb analysis. [ABSTRACT FROM AUTHOR]

Quorum-quenching catalysts are of interest for potential application as biochemical tools for interrogating interbacterial communication pathways, as antibiofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-l-homoserine lactone (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is the most efficient wild-type quorum-quenching enzymes characterized to date, with a kcat/KM value of approximately 2 × 106 M-1 s-1 for N-heptanoyl-l-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 μM for preferred substrates) compared to those of typical AHL lactonases (ca. >1 mM). X-ray crystal structures of AidC alone and with the product N-hexanoyl-l-homoserine were determined at resolutions of 1.09 and 1.67 Å, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion chromatography coupled with multiangle light scattering. The structures reveal two atypical features as compared to previously characterized AHL lactonases: a "kinked" α-helix that forms part of a closed binding pocket that provides affinity and enforces selectivity for AHL substrates and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. Implications for the catalytic mechanism of AHL lactonases are discussed. [ABSTRACT FROM AUTHOR]

This investigation reports preparation of polyfluoroacrylate/clay nanocomposite with armored and encapsulated morphology via in situ reversible addition-fragmentation chain transfer (RAFT) polymerization in miniemulsion. In this case, 2,2,3,3,4,4,4-heptafluoro butyl acrylate (HFBA) is polymerized in miniemulsion in the presence of sodium montmorillonite (NaMMT) using 2-cyanopropyl dodecyl trithiocarbonate (CPDTC) as a RAFT agent. Gel permeation chromatography (GPC) analysis shows that the polymers have controlled molecular weights as well as narrow dispersity ( Đ ≈ 1.1). The morphology of nanoclay can be tuned by varying the interaction with different functional comonomers. Nanoclay is encapsulated and intercalated in presence of poly (ethylene glycol) methyl ether methacrylate (PEGMEMA) as comonomer. Addition of 2-acryloxy ethyl trimethyl ammonium chloride (AETAC) as functional comonomer leads to exfoliation of nanoclay resulting armored morphology, as evidenced by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) analyses. Poly(2,2,3,3,4,4,4-heptafluoro butyl acrylate) (PHFBA) armored with nanoclay shows improved thermal stability and water resistance. [ABSTRACT FROM AUTHOR]

The ability of human serum albumin to capture unbound copper under different clinical conditions is an important variable potentially affecting homeostasis of this element. Here, we propose a simple procedure based on size-exclusion chromatography with on-line UV and nitrogen microwave-plasma atomic-emission spectrometry (MP-AES) for quantitative evaluation of Cu(II) binding to HSA upon its glycation in vitro. The Cu-to-protein molar ratio for non-glycated albumin was 0.98 ± 0.09; for HSA modified with glyoxal (GO), methylglyoxal (MGO), oxoacetic acid (GA), and glucose (Glc), the ratios were 1.30 ± 0.22, 0.72 ± 0.14, 0.50 ± 0.06, and 0.95 ± 0.12, respectively. The results were confirmed by using ICP-MS as an alternative detection system. A reduced ability of glycated protein to coordinate Cu(II) was associated with alteration of the N-terminal metal-binding site during incubation with MGO and GA. In contrast, glycation with GO seemed to generate new binding sites as a result of tertiary structural changes in HSA. Capillary reversed-phase liquid chromatography with electrospray-ionization quadrupole-time-of-flight tandem mass spectrometry enabled detection and identification of Cu(II) coordinated to the N-terminal metal-binding site (Cu(II)-DAHK) in all tryptic digests analyzed. This is the first report confirming Cu(II)-DAHK species in HSA by means of high-resolution tandem mass spectrometry, and the first report on the use of MP-AES in combination with chromatographic separation. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Online comprehensive two-dimensional liquid chromatography (online LCxLC) presents high peak capacity compared with one-dimensional liquid chromatography, and reasonable operation time compared with an offline or stop-flow mode. Among various combinations, coupling of two reversed-phase (RPxRP) columns generates a high peak capacity product rate. Its wide applicability made RPxRP a promising technique in separation of complex samples. This review discusses the practical considerations in development and application of an RPxRP system, including systematic investigation of column stationary phase chemistry, column combinations, mobile phase system, interface, gradient types, and achieving fast analysis in LCxLC. In addition, many efforts are given to methods that increase the fraction coverage because this is the main obstacle caused by the correlated separation mechanism in RPxRP. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

A novel collagenolytic serine protease was identified and then purified (along with ficin) to apparent homogeneity from the latex of fig ( Ficus carica , var. Brown Turkey) by two step chromatographic procedure using gel and covalent chromatography. The enzyme is a monomeric protein of molecular mass of 41 ± 9 kDa as estimated by analytical gel filtration chromatography. It is an acidic protein with a pI value of approximately 5 and optimal activity at pH 8.0–8.5 and temperature 60°C. The enzymatic activity was strongly inhibited by PMSF and Pefabloc SC, indicating that the enzyme is a serine protease. The enzyme showed specificity towards gelatin and collagen (215 GDU/mg and 24.8 CDU/mg, respectively) and non-specific protease activity (0.18 U/mg against casein). The enzyme was stable and retained full activity over a broad range of pH and temperature. The fig latex collagenolytic protease is potentially useful as a non-microbial enzyme with collagenolytic activity for various applications in the fields of biochemistry, biotechnology and medicine. [ABSTRACT FROM AUTHOR]

Octenyl-succinic anhydride (OSA)-modified starch was used as an emulsifier in oil-in-water emulsions. Multiple analytical techniques, including size-exclusion chromatography (SEC), confocal laser scanning microscopy (CLSM), and laser diffraction particle size analysis were used to determine the structure and emulsification properties of OSA-starch. The results indicated that OSA-starch with a smaller molar mass exhibited improved emulsification properties than its larger counterparts. Furthermore, samples pretreated by enzymatic modification for 25 min showed the best droplet size distribution, improved emulsion stability, and minimum aggregation. It was also determined that stable emulsions were not formed when the average molar mass of OSA-starch was too small. The emulsion ability of OSA-starch enzymatically modified for 35 min was found to be a little inferior than that of OSA-starch upon enzyme treatment for 25 min. [ABSTRACT FROM AUTHOR]

A multiple-frontal analysis (MFA) method, which was based on the data from multiple-frontal experiment, was reported to provide the most reliable adsorption parameters, particularly for the design of continuous chromatographic processes. However, if size-exclusion phenomenon occurs simultaneously with adsorption, the application of the MFA method is impracticable unless the size-exclusion factor is known beforehand. To address this issue, we developed the strategy of estimating empirically both adsorption parameter and size-exclusion factor on the basis of the multiple-frontal experimental data for a linear isotherm system. First, the existing MFA method was modified in such a way that the data related to the adsorption equilibrium of interest could be obtained without having any information on the size-exclusion factor. The result from such a modified MFA method was then applied to a series of repetitive simulations based on the reformed column model. Finally, the result from such simulation works was combined with the result from the modified MFA method, which could eventually allow the calculation of the adsorption parameter. It was confirmed from a case study that the aforementioned strategy was highly effective in estimating empirically both the adsorption parameter and the size-exclusion factor on the basis of the multiple-frontal experimental data. [ABSTRACT FROM AUTHOR]