Molecular Weight Characterization Research Articles

The New Methods for Characterization of Molecular Weight of Supramolecular Polymers

The New Methods for Characterization of Molecular Weight of Supramolecular Polymers

Advancing Molecular Weight Determination of Lignin by Multi-Angle Light Scattering.

Due to the complexity and recalcitrance of lignin, its chemical characterization is a key factor preventing the valorization of this abundant material. Multi-angle light scattering (MALS) is becoming a sought-after technique for absolute molecular weight (MW) determination of polymers and proteins. Lignin is a suitable candidate for MW determination via MALS, yet further investigation is required to confirm its absolute MW values and molecular size. Studies aiming to break down lignin into a variety of renewable products will benefit greatly from a simple and reliable determination method like MALS. Recent pioneering studies, discussed in this review, addressed several key challenges in lignin's MW characterization. Nevertheless, some lignin-specific issues still need to be considered for in-depth characterization. This study explores how MALS instrumentation manages the complexities of determining lignin's MW, e.g., with simultaneous fractionation and fluorescence interference mitigation. Additionally, we rationalize the importance of a more detailed light scattering analysis for lignin characterization, including aspects like the second virial coefficient and radius of gyration.

Polymer Characterization by Size-Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): A Tutorial Review.

This tutorial review presents the theory and application of SEC-MALS with minimal equations and a focus on synthetic polymer characterization, serving as an entry point for polymer scientists who want to learn more about SEC-MALS. We discuss the principles of static light scattering, outline its capability to generate absolute weight-average molar mass values, and extend its application to SEC-MALS. Practical elements are emphasized, enabling researchers to appreciate how values for , and are determined in an SEC-MALS experiment and how experimental conditions and input values, such as the specific refractive index increment ( ), influence the results. Several illustrative SEC-MALS experiments demonstrate the impact of separation quality on (as opposed to ), the appearance of contaminants in SEC chromatograms from sample preparation, the influence of concentration on data quality, and how polymer topology affects molecular weight characterization in SEC. Finally, we address practical considerations, common issues, and persistent misconceptions.

Production of isotopically enriched high molecular weight hyaluronic acid and characterization by solid-state NMR

Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g. 13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to 13C NMR is the low natural abundance of 13C, necessitating the generation of HMW-HA that is enriched with 13C isotopes. Here we present a convenient method to obtain 13C- and 15N-enriched HMW-HA in good yield from Streptococcus equi subsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.

Viscosity of Polymer Solutions and Molecular Weight Characterization.

Since the pioneering research by Staudinger on dilute solution viscosity and its relation to the polymer molecular weight, viscosity analysis has become a valuable technique for polymer characterization. The conventional approach is based on the Huggins approximation of the solution-specific viscosity by a quadratic function of concentration, c. We show how to reformulate this approach in a universal form by representing a solution-specific viscosity, ηsp, as a generalized universal function ηsp(c) = α(c/c*) + (1 - α)(c/c*)2 of chain overlap concentration, c*, determined at ηsp = 1, with numerical coefficients α = 0.745 ± 0.005 for good and 0.625 ± 0.008 for a θ solvent. This viscosity representation can be viewed as a calibration curve for molecular weight determination from a measurement of the solution viscosity at a given solution concentration. Furthermore, the molecular weight dependence of the overlap concentration provides a means for quantifying the polymer/solvent affinity and the solvent effect on chain flexibility. The extension of the approach to semidilute solutions opens a path for obtaining molecular weight in a broad concentration range without requiring a dilution and monitoring its change during the polymerization reaction from solution viscosity.

Quality evaluation of immunomodulatory polysaccharides from Agaricus bisporus by an integrated fingerprint technique

AbstractAgaricus bisporus (A. bisporus) is a rich source of polysaccharides with medical and nutraceutical interests; however, the complex structure of A. bisporus polysaccharides (ABPs) poses a challenge to its quality control. This study aims to investigate the immunomodulatory fractions of ABPs and further establish a quality control system for the detection and characterization of bioactive ABPs. Two fractions of ABPs were obtained using gradient alcohol precipitation combined ultrafiltration, followed by characterization of molecular weight, monosaccharide composition, chemical structure, and evaluation of immunomodulatory activity. Results showed that the two isolated fractions with different molecular weight, 8.95 × 106 Da (ABP17‐1) and 1.73 × 104 Da (ABP17‐2), were more homogeneous and exerted stronger immunomodulatory activity than that of ABPs. Furthermore, an integrated fingerprint technique including HPSEC‐MALL‐RID and HPLC was applied for the fingerprint analysis of ABPs from 17 batches of A. bisporus, which showed high similarity. Besides, two polysaccharide fractions, two monosaccharides (glucose and galactose), and the main cellulose hydrolysate glucose were identified as potential quality control markers of ABPs. Overall, this study could help better understand the structure–activity relationship of ABPs and serve as a guidance for the quality control of active ABPs and their related products.

Polymer Molecular Weight Determination via Fluorescence Lifetime.

Control of polymer molecular weight is critical for tailoring structure-function properties; however, traditional molecular weight characterization techniques have limited ability to determine the molecular weight of polymers in real time without sample removal from the reaction mixture, with spatial resolution, and of insoluble polymers. In this work, a fluorescence lifetime imaging microscopy (FLIM) method was developed that overcomes these limitations. The method is demonstrated with polynorbornene and polydicyclopentadiene, polymers derived from ruthenium-catalyzed ring-opening metathesis polymerization (ROMP). The polymer Mw, ranging from 35 to 570 kg/mol as determined by gel-permeation chromatography, was quantitatively correlated with the fluorescence lifetime. The revealed correlation then enabled time-resolved measurement of Mw during an ongoing ROMP reaction, requiring only 1 s per measurement (of a 45 μm × 45 μm polymer sample area), and provided spatial resolution, resulting in simultaneous characterization of polymer morphology. To provide the fluorescence signal, the initial reaction solutions contained a very low doping of a reactive norbornene monomer labeled with fluorescent boron dipyrromethene (BODIPY), such that 1 in every 107 monomers contained a fluorophore. The resulting FLIM visualization method enables the rapid determination of the molecular weights of growing polymers without removal from the reaction mixture and regardless of polymer solubility.

Pilot scale production and characterization of next generation high molecular weight and tense quaternary state polymerized human hemoglobin.

Polymerized human hemoglobin (PolyhHb) is being studied as a possible red blood cell (RBC) substitute for use in scenarios where blood is not available. While the oxygen (O2 ) carrying capacity of PolyhHb makes it appealing as an O2 therapeutic, the commercial PolyhHb PolyHeme® (Northfield Laboratories Inc.) was never approved for clinical use due to the presence of large quantities of low molecular weight (LMW) polymerichemoglobin (Hb) species (<500 kDa), which have been shown to elicit vasoconstriction, systemic hypertension, and oxidative tissue injury in vivo. Previous bench-top scale studies in our lab demonstrated the ability to synthesize and purify PolyhHb using a two-stage tangential flow filtrationpurification process to remove almost all undesirable Hb species (>0.2 µm and <500 kDa) in the material, to create a product that should be safer for transfusion. Therefore, to enable future large animal studies and eventual human clinical trials, PolyhHb synthesis and purification processes need to be scaled up to the pilot scale. Hence in this study, we describe the pilot scale synthesis and purification of PolyhHb. Characterization of pilot scale PolyhHb showed that PolyhHb could be successfully produced to yield biophysical properties conducive for its use as an RBC substitute. Size exclusion high performance liquid chromatography showed that pilot scale PolyhHb yielded a high molecular weight Hb polymer containing a small percentage of LMW Hb species (<500 kDa). Additionally, the auto-oxidation rate of pilot scale PolyhHb was even lower than that of previous generations of PolyhHb. Taken together, these results demonstrate that PolyhHb has the ability to be seamlessly manufactured at the pilot scale to enable future large animal studies and clinical trials.

Optimization of Soybean Protein Extraction Using By-Products from NaCl Electrolysis as an Application of the Industrial Symbiosis Concept

Defatted soybean flour is generated during the oil extraction process of soybean, and it has a protein content of ~50%. On the other hand, an alkaline solution of NaOH is produced during the electrolysis process of NaCl in a novel method used to make a potent disinfectant/antiseptic (HOCl). In the present work, we suggest using these two products to produce soy protein isolate (SPI), aiming to create an industrial symbiosis. A Box–Behnken experimental design was executed, and a surface response analysis was performed to optimize temperature, alkaline solution, and time used for SPI extraction. The SPI produced at optimal conditions was then characterized. The experimental results fit well with a second-order polynomial equation that could predict 93.15% of the variability under a combination of 70 °C, alkaline solution 3 (pH 12.68), and 44.7 min of the process. The model predicts a 49.79% extraction yield, and when tested, we obtained 48.30% within the confidence interval (46.66–52.93%). The obtained SPI was comparable in content and structure with a commercial SPI by molecular weight and molecular spectroscopy characterization. Finally, the urease activity (UA) test was negative, indicating no activity for trypsin inhibitor. Based on the functional properties, the SPI is suitable for food applications.

Low molecular weight sulfated chitosan isolation, characterization and anti-tuberculosis activity derived from Sepioteuthis lessoniana

The research focused on tuberculosis as it is one of the world's most serious health problems. The extracted chitin from the gladius of Sepioteuthis lessoniana converted into Chitosan (CH). The purified and freeze-dried CH was refined as Sulfated Chitosan (SCH). The SCH was converted into low molecular weight of SCH with various doses of Gamma Irradiation (GIR). Fluorescence characteristics of GIR-SCH and elemental analysis were confirmed. The structure and molecular weights of GIR-SCH were determined with FT-IR, NMR and MALDI–TOF/Mass Spectroscopy. 100 Gy of GIR-SCH significantly showed the minimum inhibitory concentration (MIC) against Mycobacterium smegmatis. The MIC against M. smegmatis was not affected by the varied sulfate levels in the identical molecular weight GIR-SCH. However, the lowest molecular weight GIR-SCH displayed a significantly MIC against M. smegmatis. In docking analysis, the 6ZT3 ligand had the lowest binding energy of −1.57 kcal/Mol indicating a superior binding interaction with GIR-SCH. The effect of molecular weight reduction by GIR on the anti-tuberculosis capacity of GIR SCH was investigated in this study which had antimicrobial implications.

Pharmacological Characterization of Low Molecular Weight Biased Agonists at the Follicle Stimulating Hormone Receptor.

Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet. In this context, we explored the pharmacological diversity of three benzamide and two thiazolidinone derivatives compared to FSH. Concentration/activity curves were generated for Gαs, Gαq, Gαi, β-arrestin 2 recruitment, and cAMP production, using BRET assays in living cells. ERK phosphorylation was analyzed by Western blotting, and CRE-dependent transcription was assessed using a luciferase reporter assay. All assays were done in either wild-type, Gαs or β-arrestin 1/2 CRISPR knockout HEK293 cells. Bias factors were calculated for each pair of read-outs by using the operational model. Our results show that each ligand presented a discrete pharmacological efficacy compared to FSH, ranging from super-agonist for β-arrestin 2 recruitment to pure Gαs bias. Interestingly, LMW ligands generated kinetic profiles distinct from FSH (i.e., faster, slower or transient, depending on the ligand) and correlated with CRE-dependent transcription. In addition, clear system biases were observed in cells depleted of either Gαs or β-arrestin genes. Such LMW properties are useful pharmacological tools to better dissect the multiple signaling pathways activated by FSHR and assess their relative contributions at the cellular and physio-pathological levels.

Polymer for Internal Hydrophobization of Cement-Based Materials: Design, Synthesis, and Properties.

A series of novel comb-like poly(butyl acrylate)-g-poly(dimethylaminoethyl methacrylate) (PBA-g-PDMAEMA) with different side chain lengths were designed and successfully synthesized by the “first main chain then side chain” method. Infrared Spectroscopy (IR), 1H Nuclear Magnetic Resonance (1H NMR), and gel permeation chromatography (GPC) were used for structural confirmation and molecular weight characterization. This polymer exhibited responsive behavior from hydrophilicity to hydrophobicity under the alkaline environment of cement-based materials, with the contact angle of 105.6°, a decreased evaporation rate, and a hydrophile–lipophile balance (HLB) value. A significant internal hydrophobic effect on cement mortar was shown in the water absorption rate, which decreased by 75.2%, and a dry shrinkage-reducing rate of more than 30%. Furthermore, this polymer can effectively slow the exothermic rate, reduce the heat release, and delay the exothermic peak of cement hydration. It was interesting that these properties showed a direct correlation with the side chain length of the comb polymer. The aims of this study are to provide a new avenue to synthesize polymers with the spontaneous hydrophilicity–hydrophobicity transition in the cement system, achieving excellent internal hydrophobicity of cement-based materials, and to offer a promising alternative to resist external erosion for improving the durability and service life of cement-based materials.

Molecular Weight Characterization of Conjugated Polymers Through Gel Permeation Chromatography and Static Light Scattering

Molecular weight is a key parameter of any polymer. Characterizing the molecular weight of conjugated polymers is often nontrivial due to their semiflexible backbones and poor solubilities. Perhaps the most used technique for measuring the molecular weight is gel permeation chromatography (GPC), and results are often calibrated relative to flexible polymer standards. This mismatch between chain flexibilities of samples and standards, combined with poor sample solubility in the mobile phase, leads to inaccuracies in many GPC measurements of conjugated polymers. In this work, we use a universal calibration combined with in-line concentration measurements to yield reliably accurate results for polymers of various stiffnesses. Accuracy of results is verified with absolute molecular weights obtained from static light scattering. We show that measuring the refractive index increment is key to confirm full recovery of the polymer and ensure accurate values of the absolute molecular weight from GPC.

Polysaccharide extracted from Taraxacum platycarpum root exerts immunomodulatory activity via MAPK and NF-κB pathways in RAW264.7 cells

Ethnopharmacological relevanceTaraxacum platycarpum Dahlst. (Korean dandelion) is a medicinal herb used in traditional medicine in Korea to treat various disease such as furuncles, mammitis, hepatitis, jaundice. Moreover, a decoction prepared from T. platycarpum leaves and stems is an effective treatment for cancer, glycosuria, liver disease, pleurodynia, and stomach problems. Aim of the studyThe main objective of this work was to study the composition and structural properties of polysaccharides (TPP) from Taraxacum platycarpum Dahlst. root and investigate the immunostimulatory activity on RAW264.7 cells. Materials and methodsTPP was extracted from T. platycarpum using hot water extraction, ethanol precipitation method and its fractionated using DEAE-Sepharose fast flow column. The composition, molecular weight, and structural characterization of TPP and its fractions were evaluated by various techniques. Further, the immunostimulatory activity of polysaccharides was tested on murine macrophage cell line RAW264.7 by various in vitro assays. The structure effect of TPP on RAW264.7 cells was studied by the removal of sulfate (desulfation) and protein (deproteinization) contents from TPP. ResultsWe obtained three fractions namely TPP-1, TPP-2, and TPP-3 which mainly consisted of carbohydrates (75.55, 52.71, and 48.41%), sulfate (8.42, 15.19, and 27.67%), uronic acid (1.27, 6.56, and 4.39%), and protein (8.15, 24.85, and 9.73%). The average molecular weight of the fractions was 56.7, 108.2, and 132.3 × 103 g/mol, respectively. The polysaccharides activate the RAW264.7 cell to produce a significant amount of NO and upregulate the various mRNA expression by the activation of MAPK and NF-κB pathways via TLR4, TLR2, and CR3 receptors. The structurally modified deproteinated derivative (DP-TPP-2) more effectively decreases the NO production which means the protein content of TPP-2 mainly contributes to the RAW264.7 cells activation. The structure of DP-TPP-2 primarily consists of 1 → 2)-Galp, 1 → 6)-Glup, 1 → 2) - Rhap, and 1 → 5) - Arap glycosidic linkages. ConclusionsThe present study demonstrated that the polysaccharide isolated from T. platycarpum shows admirable immunostimulatory by the activation of MAPK and NF-κB pathways through TLR4, TLR2, and CR3 receptors. The protein content of polysaccharides mainly contributes to the RAW264.7 cells activation. Our study results could be useful for developing a new immunostimulant agent.

Characterization of sarcoplasmic protein in the meat using SDS-PAGE method

A characterization of the sarcoplasmic protein in the meat had been investigated. The method that was utilized in this study was SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) which played a role in analyzing the molecular weight of the meat protein. The characterization of the molecular weight depended on the concentration of acrylamide gel that has been used. The result reported that using of 10% acrylamide gel showed difference of the molecular weight characterization from the sarcoplasmic protein. The difference of protein in the meat is on sarcoplasmic proteins. Protein with molecular weights of 50.8 kDa was not found in pork. Therefore, this molecular weight of the protein can potentially serve as a key for differentiating pork and other meats.

Part II: The influence of substrate type, film thickness, and primary ion type on molecular weight characterization of linear polydimethyl siloxane films by secondary ion mass spectrometry

In our previous paper, we showed that the molecular weight of linear, trimethyl‐terminated polydimethyl siloxane (PDMS) can be estimated using a standard curve by measuring PDMS endgroup‐related signal normalized to backbone‐related signal for PDMS films that are thick relative to the information depth of SIMS. Within industrial surface analysis laboratories, it is more likely that PDMS fluid is encountered as a thin film, perhaps as a contaminant or an additive that has exuded from a bulk material. This paper explores the influence of PDMS film thickness, substrate type, and primary ion projectile type (cluster vs. monoatomic) on the ratio of measured endgroup:backbone peak area ratio. These factors are shown to have a significant influence on the measured spectra, and these effects should be carefully considered before making molecular weight estimates of PDMS based on SIMS peak ratios.

Part I: Molecular weight characterization of linear polydimethyl siloxanes by secondary ion mass spectrometry

A correlation of SIMS ion intensities to the molecular weight of linear, trimethyl‐terminated polydimethyl siloxane (PDMS) has been developed by normalizing PDMS endgroup‐related signal to backbone‐related signal for a set of well‐characterized polymers with narrow molecular weight distributions. These initial experiments on thick PDMS films illustrate that PDMS molecular weight can be estimated using SIMS. A second paper describes the challenges in analyzing thin PDMS films on metal and polymer substrates. PDMS molecular weight determination is important to understanding surface‐related performance of materials using silicone additives across many different platforms including coefficient of friction, slip, mar, mold release, and nonstick properties as well as providing more specific characterization of surface contamination.

Characterization of Molecular Weight–Based Fluorescent Organic Matter and Its Removal in Combination of Constructed Wetland with Activated Sludge Process

Integrated constructed wetland into activated sludge process has a potential in improving treated wastewater with high organic loading. However, biological activities on those processes will generate microbial by-products from substrate metabolism and cell lysis. The presence of those compounds in effluent of wastewater treatment causes problems in source water. This study combines fluorescence excitation emission matrices (FEEM) with high-performance size exclusion chromatography with fluorescence detector (HPSEC-FLD) to characterize molecular weight–based fluorescent of organic matter and its removal in combination of constructed wetland with activated sludge process. The results show that three components of fluorescence organic: fulvic acid–like (Ex/Em 250/440 nm), SMP-like (Ex/Em 280/350 nm), humic acid–like (Ex/Em 340/420 nm), have been identified in all samples by the FEEM. Further, the HPSEC-FLD, which was set up based on chosen fluorescence wavelength, revealed two different apparent molecular weight (AMW) fractions: high molecular weight (HMW)/biopolymer (50,000 Da) and medium molecular weight (MMW)/humic substance–like (3000–650 Da). Peak-fitting determines that the area of MMW is higher than the area of HMW of all fluorescence organic components, and the area of HMW of fluorescence fulvic acid–like is comparable with the area of SMP-like, and no HMW of humic acid–like detected. Humic acid–like and fulvic acid–like were removed during treatment, while metabolite by-product were released as shown by increasing fluorescence SMP-like and TOC concentration. This method gives new insight to characterize organic matter for assessing effluent of wastewater quality and determining the appropriate water treatment.

New series of metal complexes by amphiphilic biopolymeric Schiff bases from modified chitosans: Preparation, characterization and effect of molecular weight on its biological applications

To improve biological activity of chitosans, new Zn(II), Pd(II) and Pt(II) complexes with biopolymeric amphiphilic Schiff bases anchored in different molecular weight chitosans matrices modified with salicylaldehyde and glycidol were prepared. Salicylaldehyde was introduced to generate complexing Schiff base sites in the chitosans matrix while glycidol is intended to increase the water solubility of the resulting biopolymeric complexes. These novel complexes were characterized using various techniques and assayed for antimicrobial and antitumor activity. The effectiveness of modification was evaluated using FTIR spectroscopy, and thermal behavior of the complexes by TG/DTG-DTA. XPRD showed that the crystallinity of the ligand diminished after the metal complexation. Surface morphologies, investigated by SEM, revealed that the complexes are rougher than chitosan matrix, and the presence of metallic ions was confirmed by EDX. Electronic spectra suggested square planar geometry for Pd(II) and Pt(II) complexes. Concerning antimicrobial activity, the novel complexes exhibited higher antibacterial efficiency against Pseudomonas syringae than against the Fusarium graminearum fungi regarding the free ligand. Complexes also exhibited high antitumor effects against the MCF-7 breast cancer cells, with certain selectivity regarding non-tumor cells (Balb/C 3T3 clone A31) depending on concentration and molar mass, indicating that they could potentially be used for antitumor applications.

Characterization of bioactive and nutraceutical compounds occurring in olive oil processing wastes.

Several bioactive compounds, including phenolic acids and secoiridoids, are transferred from olive drupes to olive oil during the first stage of production. Here, the characterization of these low molecular weight (LMW) compounds in olive oil and in closely related processing materials, like olive leaves (OL) and olive mill wastewaters (OMW), was faced up, for the first time, by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS). A novel binary matrix composed of 1,8-bis(tetramethylguanidino)naphthalene (TMGN) and 9-aminoacridine (9AA) (1:1 molar ratio), displaying excellent ionization properties at low levels of laser energy, was employed in reflectron negative ion mode by a MALDI TOF/TOF system equipped with a neodymium-doped yttrium lithium fluoride (Nd:YLF) laser (345 nm). MS/MS experiments were performed by using ambient air as the collision gas. Four major secoiridoids typically present in olive oil, i.e., the aglycones of oleuropein and ligstroside, and oleacein and olecanthal at m/z 377.1, 361.1, 319.1 and 303.1, respectively, were detected in virgin olive oil (VOO) extracts, along with some of their chemical/enzymatic hydrolysis by-products, such as elenolic (m/z 241.1), decarboxymethyl-elenolic acids (m/z 183.1) and hydroxytyrosol (m/z 153.1). Besides oleuropein aglycone and oleacein, hydroxylated derivatives of decarboxymethyl-elenolic acid and hydroxytyrosol were evidenced in OMW. While oleuropein was confirmed in OL extracts, several interesting phenolic compounds, including hydroxytyrosol, were recognized in OMW. The proposed approach based on the use of a novel binary matrix for MALDI MS/MS analyses of LMW bioactive compounds can be considered a promising analytical tool for a rapid screening of the phenolic fraction in olive oils and related processing wastes.