Size-exclusion Chromatography With Multi-angle Laser Light Scattering Research Articles

Towards a better understanding of synergistic enzyme effects during refining of cellulose fibers

Refining of cellulose fibers is essential for reaching desired paper properties, yet highly energy demanding. Enzymes like endoglucanases (e.g. EndoC) are increasingly used to reduce energy consumption during pulp refining. However, prediction of the enzyme effect is still a major concern, considering the high variety of commercially available enzyme formulations, containing a range of different enzymes. In this study, synergisms of xylanases and β-glucosidases in combination with endoglucanases purified from enzyme formulations were studied and related to their refining performance. Size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) of carboxymethylcellulose revealed that a narrow size distribution and a high reduction in molecular weight are beneficial characteristics for refining. SEC-MALLS of hardwood pulp resulted in pronounced formation of low molecular weight fractions (log MW 4.3) for most efficient refining enzymes. Application of enzyme formulations and combinations of endoglucanase EndoC with β-glucosidase or xylanase using Fourier-transform infrared spectroscopy (FTIR) revealed synergistic effects that promoted degradation of amorphous parts of cellulose. Laboratory refining trials on hardwood pulp confirmed the increase in degree of refining and tensile index after addition of xylanase and β-glucosidase. Surface plasmon resonance (SPR) analysis resulted in strong binding of endoglucanases to regenerated cellulose, which correlated to refining performance.

Effects of Drug Physicochemical Properties on In-Situ Forming Implant Polymer Degradation and Drug Release Kinetics.

In-situ forming implants (ISFIs) represent a simple, tunable, and biodegradable polymer-based platform for long-acting drug delivery. However, drugs with different physicochemical properties and physical states in the polymer-solvent system exhibit different drug release kinetics. Although a few limited studies have been performed attempting to elucidate these effects, a large, systematic study has not been performed until now. The purpose of this study was to characterize the in vitro drug release of 12 different small molecule drugs with differing logP and pKa values from ISFIs. Drug release was compared with polymer degradation as measured by lactic acid (LA) release and change in poly(DL-lactide-co-glycolide) (PLGA) molecular weight (MW) measured by size exclusion chromatography with multi-angle laser light scattering (SEC-MALS). Drug physical state and morphology were also measured using differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Together, these results demonstrated that hydrophilic drugs have higher burst release at 24 h (22.8–68.4%) and complete drug release within 60 days, while hydrophobic drugs have lower burst release at 24 h (1.8–18.9%) and can sustain drug release over 60–285 days. Overall, drug logP and drug physical state in the polymer–solvent system are the most important factors when predicting the drug release rate in an ISFI for small-molecule drugs. Hydrophilic drugs exhibit high initial burst and less sustained release due to their miscibility with the aqueous phase, while hydrophobic drugs have lower initial burst and more sustained release due to their affinity for the hydrophobic PLGA. Additionally, while hydrophilic drugs seem to accelerate the degradation of PLGA, hydrophobic drugs on the other hand seem to slow down the PLGA degradation process compared with placebo ISFIs. Furthermore, drugs that were in a crystalline state within the ISFI drugs exhibited more sustained release compared with amorphous drugs.

Determination of the Molecular Mass of Membrane Proteins Using Size-Exclusion Chromatography with Multiangle Laser Light Scattering (SEC-MALLS ).

Size-exclusion chromatography coupled to multiangle laser light scattering (SEC-MALLS) is the perfect method to determine the oligomeric state of membrane proteins as this method works in solution and is totally independent from prior assumptions such as detergent-to-protein ratio or the shape of the protein. In a relatively short time (ca. 30min), the molecular mass and quality of a membrane protein preparation can be determined. Here, I provide a detailed protocol on how to perform a SEC-MALLS run and show exemplary chromatograms and their analysis.

Determination of the absolute molar mass of acetylated eucalyptus kraft lignin by two types of size-exclusion chromatography combined with multi-angle laser light-scattering detectors

Abstract The molar mass (MM) and polydispersity indices (PDI) of acetylated hardwood kraft lignin (Ac-HWKL) and Ac-HWKL fractions were measured by size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS). The detectors worked at LL658 nm and LL785 nm. The MM of Ac-HWKL measured at 785 nm was much smaller than that measured at 658 nm. The number-average (Mn) and weight-average (Mw) molar masses of Ac-HWKL determined at 785 nm were approximately 6.2 and 6.5 times larger, respectively, than the values estimated using a conventional calibration curve created with authentic polystyrene standards in tetrahydrofuran (THF). Based on the Mw of Ac-HWKL fractions, the Mark-Houwink-Sakurada equation for Ac-HWKL was established to be [η]/ml g−1=0.320 M0.24 in THF and [η]/ml g−1=0.142 M0.26 in dimethyl sulfoxide (DMSO). These results demonstrate that Ac-HWKL has a more compact structure in an organic solvent than polystyrene.

Solution-state structures of the cellulose model pullulan in lithium chloride/N,N-dimethylacetamide

Pullulan is soluble in both N,N-dimethylacetamide (DMAc) and LiCl/DMAc. Here, the solution-state structures of pullulan in these two solvents were studied by size-exclusion chromatography with multi-angle laser light scattering (SEC/MALLS) and nuclear magnetic resonance (NMR) analyses. SEC/MALLS and off-line refractive index analyses revealed that the hydroxy groups of pullulan interact with LiCl in DMAc, as observed previously for cellulose. The nine different COH protons of pullulan in DMAc and LiCl/DMAc appeared independently in NMR spectra and were successfully assigned. When LiCl was present, the degree of dissociation of all COH protons of pullulan was increased by Li+ and Cl− ions, and the conformations of the COH groups became stable and restricted in DMAc. Based on the obtained results, the solution-state structures of cellulose in LiCl/DMAc are hypothesized to contain similar interactions between the cellulose hydroxy groups, and Li+ and Cl− in LiCl/DMAc to those observed for pullulan.

Structure of the Branched-chain Amino Acid and GTP-sensing Global Regulator, CodY, from Bacillus subtilis

CodY is a branched-chain amino acid (BCAA) and GTP sensor and a global regulator of transcription in low G + C Gram-positive bacteria. It controls the expression of over 100 genes and operons, principally by repressing during growth genes whose products are required for adaptations to nutrient limitation. However, the mechanism by which BCAA binding regulates transcriptional changes is not clear. It is known that CodY consists of a GAF (cGMP-stimulated phosphodiesterases, adenylate cyclases, FhlA) domain that binds BCAAs and a winged helix-turn-helix (wHTH) domain that binds to DNA, but the way in which these domains interact and the structural basis of the BCAA dependence of this interaction are unknown. To gain new insights, we determined the crystal structure of unliganded CodY from Bacillus subtilis revealing a 10-turn α-helix linking otherwise discrete GAF and wHTH domains. The structure of CodY in complex with isoleucine revealed a reorganized GAF domain. In both complexes CodY was tetrameric. Size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) experiments showed that CodY is a dimer at concentrations found in bacterial cells. Comparison of structures of dimers of unliganded CodY and CodY-Ile derived from the tetramers showed a splaying of the wHTH domains when Ile was bound; splaying is likely to account for the increased affinity of Ile-bound CodY for DNA. Electrophoretic mobility shift and SEC-MALLS analyses of CodY binding to 19–36-bp operator fragments are consistent with isoleucine-dependent binding of two CodY dimers per duplex. The implications of these observations for effector control of CodY activity are discussed.

Preparation and extensive characterization of hyaluronan with narrow molecular weight distribution

The physicochemical properties and biological functions of hyaluronan (HA) are closely related to its molecular weight (MW) and molecular weight distribution (MWD). Therefore, it is crucially important to provide a reliable characterization of these parameters for proper use of HA and its degradation products in both chemical and clinical fields. In this study, we present a novel method for the preparation of HA fragments of defined size with narrow molecular weight distribution. The HA fractionation was performed using an anion-exchange chromatography and is applicable either after enzymatic or chemical hydrolysis of polymeric HA. Isolated fractions with a molecular weight ranging from 3000–420,000gmol−1 were analyzed by size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS). Hundred-milligram scale HA fragments were obtained from 5g hyaluronan starting material. Independently on weight-average molecular weight (Mw), the polydispersity index (PDI) of the HA fractions was less than 1.23. The fractionation methodology can be easily up-scaled and is applicable on any negatively charged polymers. We have also found that PDI is insufficient to characterize almost monodisperse fractions and for proper material characterization we proposed a new characteristic termed “distribution angle”, calculated from the slope of the cumulative molecular weight distribution curve. Compared to PDI, the distribution angle reflects more efficiently changes in size distribution and thus is highly recommended to be used along with Mw determination of any polymer.

Effects of ultrasound on molecular properties, structure, chain conformation and degradation kinetics of carboxylic curdlan

In this study, high-intensity ultrasound (20 kHz), a simple, effective and without any additive method, was used to the degradation of carboxylic curdlan (Cc) produced by 4-acetamido-TEMPO-mediated oxidation. The effects of ultrasound on molecular properties, structure and chain conformations of Cc were investigated by viscometry, size-exclusion chromatography with multiangle laser-light scattering (SEC-MALLS) analysis, as well as FTIR and NMR spectroscopies. The results indicated that the intrinsic viscosity [η] and the weight-average molecular weight (Mw) of Cc decreased obviously after ultrasound, and a uniform and narrow distribution of degradation product was obtained. The z-average radius of gyrations (Rg) firstly increased and then decreased as the sonication time prolonged. Ultrasound destroyed the hydrogen bonds resulting in the transition from compact random coil conformation to more flexible and even shorter extended chains. Ultrasonic treatment could not alter the primary chemical structure of Cc molecules according to the structural analysis by FTIR and NMR spectroscopies. Degradation kinetics based on Schmid model was applied to estimate the degradation rate constant k. It was found that the k value of Cc decreased with increasing the polymer concentration from 0.05 to 0.2% (w/v).

Structural characterization and antioxidant activity of a heteropolysaccharide isolated from Hedysarum polybotrys

A water-soluble polysaccharide (HPS3aS) with a molecular mass of 1.22 × 104 Da was isolated from Hedysarum polybotrys using anion-exchange and gel-permeation chromatography. HPS3aS exhibits a globular-shaped conformation in 0.1 M NaNO3 by size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS). The investigation of the structural features of this heteropolysaccharide through a combination of chemical and instrumental analyses revealed that the backbone of HPS3aS is composed of α-d-(1 → 4)-linked glucopyranose residues, which occasionally branched at O-6. The branches are composed of (1 → 4)-linked galactopyranose residues and terminated with glucopyranose residues. HPS3aS possesses good in vitro antioxidant activity, as evaluated by scavenging assays with 1,1-diphenyl-2-picrylhydrazyl, hydroxyl, and superoxide radicals, which suggests that HPS3aS could be a potential antioxidant.

Structure of the Phosphatase Domain of the Cell Fate Determinant SpoIIE from Bacillus subtilis

Sporulation in Bacillus subtilis begins with an asymmetric cell division producing two genetically identical cells with different fates. SpoIIE is a membrane protein that localizes to the polar cell division sites where it causes FtsZ to relocate from mid-cell to form polar Z-rings. Following polar septation, SpoIIE establishes compartment-specific gene expression in the smaller forespore cell by dephosphorylating the anti-sigma factor antagonist SpoIIAA, leading to the release of the RNA polymerase sigma factor σF from an inhibitory complex with the anti-sigma factor SpoIIAB. SpoIIE therefore couples morphological development to differential gene expression. Here, we determined the crystal structure of the phosphatase domain of SpoIIE to 2.6 Å spacing, revealing a domain-swapped dimer. SEC-MALLS (size-exclusion chromatography with multi-angle laser light scattering) analysis however suggested a monomer as the principal form in solution. A model for the monomer was derived from the domain-swapped dimer in which 2 five-stranded β-sheets are packed against one another and flanked by α-helices in an αββα arrangement reminiscent of other PP2C-type phosphatases. A flap region that controls access of substrates to the active site in other PP2C phosphatases is diminished in SpoIIE, and this observation correlates with the presence of a single manganese ion in the active site of SpoIIE in contrast to the two or three metal ions present in other PP2C enzymes. Mapping of a catalogue of mutational data onto the structure shows a clustering of sites whose point mutation interferes with the proper coupling of asymmetric septum formation to sigma factor activation and identifies a surface involved in intramolecular signaling.

Ring-opening polymerization of γ-glycidoxypropyltrimethoxysilane catalyzed by multi-metal cyanide catalyst

Ring-opening polymerizations of γ-glycidoxypropyltrimethoxysilane (GPTMS) were carried out by using multi-metal cyanide (MMC) catalyst and the synthesized homopolymer was a comb-shaped polymer with regular structure. The structure of the polymer obtained (P-GPTMS) was characterized by FTIR, 1H-NMR, and 29Si-NMR spectroscopy, and the molecular weight and its distribution were analyzed by size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS). The thermal behavior of P-GPTMS was investigated by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). P-GPTMS with high molecular weight (Mn = 18,000–80,000) and narrow molecular weight distribution (1.10–1.35) were synthesized when the dosage of MMC catalyst was 0.1% and polymerization temperature was 130 °C. The molecular weight of the product could be adjusted by controlling the polymerization time. The Tg of P-GPTMS is in the range of −34 to −30 °C. On the basis of the TGA data, the decomposition rate of P-GPTMS reached its peak at 374.14 °C and the entire decomposition stopped at 600 °C.

Determination of molecular weight of polyethylene glycol using size-exclusion chromatography with multi-angle laser light scattering and acid–base titration

Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) and acid–base titration were used to determine the molecular weight of a polyethylene glycol with low molecular weight. All potential uncertainty factors for each measurement were evaluated using cause-effect diagrams. Weight analysis was applied to harmonize the two different measurement results. It was found that the main uncertainty sources of SEC-MALLS measurement arose from the d n/d c value, the calibration constant of the differential refractive index (DRI) detector, and the intermediate precision of the instrument, while the sample mass, the titration volume and the concentration of titrant contributed to the uncertainty of titration measurement. The weighted mean value of the two measurement results was taken as the molecular weight of the polyethylene glycol.

Improved reproducibility in the determination of the molecular weight of chitosan by analytical size exclusion chromatography

The reproducibility of the determination of the molecular weight of chitosans in the 90–210 kDa range ( M n) by analytical size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) was improved by reducing the salt concentration in the mobile phase from (0.3 M acetic acid, 0.2 M sodium acetate, and 0.8 mM sodium azide) to (0.15 M acetic acid, 0.1 M sodium acetate, and 0.4 mM sodium azide) using Tosoh TSKgel G6000PW XL and G5000PW XL columns in series. The variability of measured molecular weight was significantly reduced by lowering the acetate concentration in the mobile phase, while the average molecular weight did not change significantly. The coefficient of variation of the number-average molecular weight, CV( M n), decreased from 7–12% to 3–6% upon mobile phase dilution. This reduced variability in molecular weight of chitosans obtained from SEC is a significant improvement when precise values of chitosan molecular weight are required, for example in stability studies where viscosity changes in concentrated chitosan solutions are assessed, and in gene delivery applications.

Corona-Stabilized Interpolyelectrolyte Complexes of SiRNA with Nonimmunogenic, Hydrophilic/Cationic Block Copolymers Prepared by Aqueous RAFT Polymerization

The complexation of small interfering ribonucleic acid (siRNA) with a series of specifically designed block copolymers consisting of the hydrophilic, nonimmunogenic monomer N-(2-hydroxypropyl)methacrylamide (HPMA) and the cationic monomer N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) has been investigated for potential siRNA stabilization and delivery applications. Specific compositions of poly(HPMA-b-DMAPMA) copolymers were synthesized via aqueous reversible addition−fragmentation chain transfer (RAFT) polymerization and characterized using aqueous size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) and 1H NMR spectroscopy. The degree of soluble complex formation between a model siRNA and the polymers was determined by centrifugal membrane filtration experiments and quantitated by scintillation counting of 32P ATP-labeled siRNA to determine complex solubility and to estimate the degree of complexation relative to cationic and neutral block lengths. Dynamic and static light scattering methods were employed to determine the hydrodynamic radii, molecular weights, and second virial coefficients of the complexes and to demonstrate their unimodal size distributions. In vitro enzymatic degradation studies of selected siRNA/block copolymer complexes were conducted to demonstrate the enhanced stability of the siRNA/poly(HPMA-b-DMAPMA) complexes. Furthermore, the siRNA/polymer complexes dissociate slowly under gel electrophoresis conditions. Therefore, the siRNA/polymer complexes demonstrate some highly desirable properties for potential applications in therapeutic siRNA stabilization and delivery.

Efficient Divergent Synthesis of Dendronized Polymers with Extremely High Molecular Weight: Structural Characterization by SEC-MALLS and SFM and Novel Organic Gelation Behavior

High molecular weight poly(p-hydroxystyrene) (PHS) was dendronized in high yield by the divergent grafting of aliphatic polyester dendrons using acetal and ketal protected anhydride derivatives of bis(hydroxymethyl)propionic acid. Relatively low polydispersities (PDI = 1.1-1.3) were maintained throughout the dendronization process. Detailed characterization of a series of these polymers by size exclusion chromatography (SEC) and SEC with multi-angle laser light scattering (SEC-MALLS) clearly shows the effect of dendron steric bulk on the persistence length (ξ) of the polymer main chain in solution. As a result of the fixed degree of polymerization (DP), the main chain rigidification that accompanies lateral dendron growth causes an unusual relationship between R g,z and molecular weight across a series of generations. The rodlike structure and contour length of the alkylated dendronized polymers was also characterized by scanning force microscopy (SFM) on mica, highly oriented pyrolytic graphite (HOPG), and molybdenum disulfide (MoS 2 ) surfaces. One of the G3 dendronized polymers with an alkylated periphery showed significant physical gelation behavior at concentrations below 1 wt % in several organic solvents, which constitutes additional evidence that the solution phase rigidity of a polymer chain can be enhanced by dendronization.

Film Properties of Hydroxypropyl Starch

AbstractThe present study describes the preparation of low‐substituted hydroxypropyl (HP) starches and investigation of their suitability for the development of flexible, highly transparent cast films. Changes in molecular composition due to processing conditions were identified by determining molar mass distribution. Molecular characterisation was performed both by static light scattering (SLS) and size‐exclusion chromatography with multi‐angle laser light scattering (SEC‐MALLS) detection. The weight‐average molar mass, mean square radius of gyration, second virial coefficient, and molar mass distribution were determined. Rheological characterisation of concentrated aqueous solutions included a study of flow behaviour as a function of shear rate and oscillatory measurement of viscoelastic properties. Significant differences in rheological properties due to hydroxypropylation conditions were established. Mechanical testing was carried out by measuring tensile strength and elongation and determining the elastic modulus. The tensile strength of cast films based on hydroxypropyl starches was 40–50 MPa. Elongation values were dependent on the type of starch, use of plasticiser and storage conditions.

Improved SDS-PAGE Molecular Weight Determination of a Succinylated Limed Ossein Gelatin

Solutions of succinylated gelatin are administered intravenously to patients as a substitute for blood plasma, and the weight-average molecular weight (M w )of the succinylated gelatin is a key parameter that governs the intravascular persistence and its clinical effectiveness as a plasma substitute. However, existing methods for determining M w values and its distribution for succinylated gelatin yield different answers, as their calibration procedures vary. In this study, SEC/MALLS (size exclusion chromatography with multiangle laser light scattering), a technique that is independent of external calibrants, was used to verify the suitability of two potential SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) calibration standards (i.e., globular proteins and cyanogen bromide cleaved collagen peptides). It was concluded that both globular proteins and collagen CNBr peptides were unsuitable as calibrants for molecular weight determination of succinylated gelatin by SDS-PAGE. The effect of both chemical composition and structure of proteins on separation by SDS-PAGE suggests that more suitable calibration standards would be those derived from the parent succinylated gelatin.