Size exclusion chromatographic characterization of sodium hyaluronate fractions prepared by high energetic sonication

Summary A high molecular weight sodium hyaluronate isolated from rooster combs was degraded by ultrasonication. High-performance size exclusion chromatography allowed rapid and accurate determination of molecular weight parameters (M_z, M_w, M_n) and distributions. The time dependence of hyaluronan ultrasonication to the molecular characteristics of the polymer was investigated. A non-random nature of the degradation process was demonstrated and the reciprocal M_n value was found to be linearly proportional to the time of ultrasonication.     

Molecular weight effect on the photoinduced birefringence and surface relief gratings formation of a methacrylate azopolymer

Samples of a methacrylic side-chain azopolymer were synthesized by a radical copolymerization of methyl-methacrylate with the methacrylic derivative of the commercial dye Disperse Red-13 (DR13). Copolymers with different molecular weight were obtained by varying the reaction time and the concentration of initiator. Samples with molecular weight averages (M_w) from 3 to 8 × 10⁴ g/mol and 1.4-1.7 polydispersity were obtained. The glass transition temperature of the samples increased linearly from the lower to the highest molecular weight. Optical quality cast films were prepared for all samples and photoinduced birefringence was successfully carried out in all samples as well as surface relief gratings could be inscribed in the cast films.     

Depolymerization study of sodium hyaluronate by flow field-flow fractionation/multiangle light scattering

Thermal depolymerization of ultrahigh-molecular-weight (UHMW) sodium hyaluronate (NaHA) was studied systematically by using frit-inlet asymmetrical flow field-flow fractionation/multiangle light scattering/differential refractive index (FI-AFlFFF/MALS/DRI). FI-AFlFFF was utilized for the size separation of NaHA samples which had been thermally degraded for varied treatment times, followed by light-scattering detection to determine MW and structural information of degraded NaHA products. Analysis of NaHA products showed time-dependent depolymerization of raw molecules into smaller-MW components, as well as unfolding of compact structures of UHMW NaHA. To determine whether the observed decrease in MW of sodium hyaluronate originated from the chain degradation of UHMW molecules or from dissociation of entangled complex particles that may have been formed by intermolecular association, narrow size fractions (1 × 10⁷–6 × 10⁷ and >6 × 10⁷ MW) of NaHA molecules were collected during FlFFF separation and followed by thermal treatment. Subsequent FI-AFlFFF/MALS analysis of collected fractions after thermal treatment suggested that the ultrahigh-MW region (>10⁷ Da) of NaHA is likely to result from supermolecular structures formed by aggregation of large molecules.     

On the action of ozone at high concentration on various grades of polyethylene and certain straight chain paraffins

The surface functionalisation of UHMWPE (ultra high molecular weight polyethylene) was successfully achieved by the action of ozone (10% by weight in oxygen) under mild conditions. The kinetics of the gas-solid reaction between O₃ and UHMWPE in powder form were measured in an IR gas cell and the pseudo-first-order rate constant was k′_UHMWPE = 1.9 × 10⁻⁴ s⁻¹. The resulting surface-oxidized UHMWPE was studied by FT-IR spectroscopy and the nature of the surface functionalities was determined. Furthermore, the surface-oxidized UHMWPE was studied by DSC (differential scanning calorimetry). It was evident that ozone attacks and oxidizes the amorphous phase of UHMWPE preserving the crystalline phase because after the ozone treatment there was an increase in the % of crystallinity. Two other polyethylene grades having respectively M_w = 15,000 Da (defined as LMWPE = low molecular weight polyethylene) and M_w = 4000 Da (defined as VLMWPE = very low molecular weight polyethylene) were studied as model compounds in comparison to UHMWPE in their reaction with ozone. Commercial liquid paraffin and n-dodecane were used as model compounds to study the reaction between high ozone concentration and alkanes.     

Controlled radical polymerization of a trialkylsilyl methacrylate by reversible addition–fragmentation chain transfer polymerization

The reversible addition–fragmentation chain transfer (RAFT) polymerization of a hydrolyzable monomer (tert‐butyldimethylsilyl methacrylate) with cumyl dithiobenzoate and 2‐cyanoprop‐2‐yl dithiobenzoate as chain‐transfer agents was studied in toluene solutions at 70 °C. The resulting homopolymers had low polydispersity (polydispersity index < 1.3) up to 96% monomer conversion with molecular weights at high conversions close to the theoretical prediction. The profiles of the number‐average molecular weight versus the conversion revealed controlled polymerization features with chain‐transfer constants expected between 1.0 and 10. A series of poly(tert‐butyldimethylsilyl methacrylate)s were synthesized over the molecular weight range of 1.0 × 10⁴ to 3.0 × 10⁴, as determined by size exclusion chromatography. As strong differences of hydrodynamic volumes in tetrahydrofuran between poly(methyl methacrylate), polystyrene standards, and poly(tert‐butyldimethylsilyl methacrylate) were observed, true molecular weights were obtained from a light scattering detector equipped in a triple‐detector size exclusion chromatograph. The Mark–Houwink–Sakurada parameters for poly(tert‐butyldimethylsilyl methacrylate) were assessed to obtain directly true molecular weight values from size exclusion chromatography with universal calibration. In addition, a RAFT agent efficiency above 94% was confirmed at high conversions by both light scattering detection and ¹H NMR spectroscopy. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 5680–5689, 2005     

Gamma radiation induced synthesis of gold nanoparticles in aqueous polyvinyl pyrrolidone solution and its application for hydrogen peroxide estimation

Gold nanoparticles (Au nps) have been synthesized in aqueous solution of polyvinyl pyrrolidone (PVP) by gamma radiolysis from HAuCl₄·3H₂O precursor and in presence of small concentrations of Ag⁺, 2-propanol and acetone. The effect of different experimental parameters, such as concentration of reactant, molecular weight of PVP on nanoparticle formation was studied. TEM image confirmed that spherical Au nps were formed when PVP of molecular weight 360,000 Da was used as capping agent. H₂O₂ is a reactant in the enzyme catalyzed reaction of o-phenylene diamine (o-PDA). The reaction product has a weak absorption in the yellow region of the spectrum. When this product interacts with Au nps, it leads to enhancement of the absorption peak. The nanoparticles synthesized by radiation method were used for estimation of H₂O₂. The absorbance value of this peak at λ_max was observed to change with H₂O₂ concentration, which was monitored for estimation of H₂O₂. The response is linear in the range of 2.5×10⁻⁶ mol dm⁻³ to 2×10⁻⁴ mol dm⁻³ and 1×10⁻⁷ mol dm⁻³ to 3×10⁻⁶ mol dm⁻³ H₂O₂ in two separate sets of experimental parameters with detection limit 1×10⁻⁷ mol dm⁻³.     

Characterization of sodium hyaluronate blends using frit inlet asymmetrical flow field-flow fractionation and multiangle light scattering

We characterized ultrahigh molecular weight sodium hyaluronate (NaHA) and blended pharmaceutical products containing NaHA using flow field-flow fractionation and multiangle light scattering–differential refractive index (FlFFF-MALS-DRI). NaHA is a water-soluble polysaccharide with a range of molecular weights (MW; 10⁵~10⁸ Da) that is found in body fluids and tissues. NaHA is also used commercially in pharmaceutical and cosmetic applications. We used a frit inlet asymmetrical FlFFF channel to separate aqueous polymers according to their hydrodynamic size, and we used on-line measurements of light scattering to obtain the MW distribution (MWD) as well as structural information about NaHA in aqueous solution. In this study, we investigated NaHA and anti-adhesive blend mixtures of NaHA (a commercial NaHA blend mixture containing sodium carboxymethyl cellulose and a new blend with hydroxyethyl starch (HES)) to determine the molecular weight distribution MWD of NaHA and the blend mixtures and to obtain structural information about these compounds in aqueous solution. We also examined the characteristics of NaHA–HES–polylactic-co-glycolic acid film products exposed to gamma radiation for sterilization purposes.     

The vaporization of semi-volatile compounds during tobacco pyrolysis

Pyrolysis of tobacco, a complex biomass matrix, was investigated to further understand thermal decomposition processes that are accompanied by evaporation of relatively stable non-polymeric endogenous compounds. Pyrolysis of two types of tobacco, bright and burley were studied using thermo-gravimetry mass spectrometry (TG–MS) and field ionization mass spectrometry (FIMS) analyses. Tobacco contains biopolymers and many non-polymeric compounds. Unlike many biomass pyrolysis tars derived from wood or cellulose, tobacco pyrolysis tars can contain significant amounts of high molecular weight endogenous constituents such as waxes and terpenes that are transferred intact. The phenomenon of evaporation of high molecular weight non-polymeric compounds is illustrated by tobacco micro-sample pyrolysis in FIMS under vacuum (at a pressure of 10⁻⁴ Torr). These experiments indicate that the evaporation of relatively stable high molecular weight species occurs below about 220 °C generating 300 Da and higher molecular weight products; and, decomposition of tobacco biopolymers such as starch, cellulose, hemicellulose, lignin, and pectin occurs mostly at temperatures higher than 220 °C producing species mostly with molecular weight below 300 Da. Some of the high molecular weight compounds, such as stigmasterol (412 Da), α-tocopherol (430 Da), and solanesol (630 Da), were tentatively identified using the FIMS spectra.     

Multidisciplinary characterization of novel emulsion polymers

A series of novel emulsion styrene‐butadiene copolymer blends were characterized using a multidisciplinary approach involving both analytical and rheological measurements. The blends were composed of 50/50 w/w high molecular weight (ca. 800,000 Da) ESBR and low molecular weight (ca. 200,000 Da) ESBR, each component having a different bound styrene level. When the difference in bound styrene between the two components was greater than 18%, a two phase co‐continuous morphology was observed by scanning probe microscopy, consistent with two glass transitions measured by temperature modulated DSC. Molecular weight and molecular weight distributions were characterized by both size exclusion chromatography and thermal field flow fractionation with multiangle light scattering detection. ThFFF was unique in its ability to detect ultra‐high molecular weight (> 10⁷ Da) fractions suggesting that traditional SEC often under‐estimates polymer molecular weight. Blending polymers of different molecular weights and styrene levels resulted in reduced molecular weight between entanglements which, based on rheological measurements, would be expected to improve processability.     

DETERMINATION OF BRANCHING DISTRIBUTION IN HIGH cis-1,4-POLYBUTADIENE BY GPC AND AUTOMATIC VISCOMETRY

Based on the methods reported by Ambler and Kraus, a method has been developed for the determination of long-chain branching distribution in polymers by the combined use of GPC and intrinsic viscosity data of polymer fractions. In this method, g_i, λ_i, G_i, m_i, the weight percentage of polymer that is branched, etc. can be used simultaneously to characterize the distribution, degree and content of branching in polymers. Some relations between molecular weight polydispersity and branching polydispersity in Nickel-based high cis-1,4-polybutadiene samples are discussed. It was found that the number of long branches λ. per unit molecular weight is a function of molecular weight and all of the samples are highly branched at a molecular weight of about 10~6.     

Contribution of Glycerol on Production of Poly(γ-Glutamic Acid) in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> NX-2

Glycerol would stimulate the production of poly(γ-glutamic acid) (γ-PGA) and decrease its molecular weight in Bacillus subtilis NX-2. When 20 g/l glycerol was added in the medium, the yield of γ-PGA increased from 26.7 ± 1.0 to 31.7 ± 1.3 g/l, and molecular weight of γ-PGA decreased from 2.43 ± 0.07 × 10⁶ to 1.86 ± 0.06 × 10⁶ Da. In addition, it was found that the decrease of γ-PGA chain length by glycerol would lead to the decrease of broth viscosity during the fermentation and enhanced the uptake of substrates, which could not only improve cell growth but also stimulate γ-PGA production. Moreover, it was also found that glycerol could effectively regulate molecular weight between 2.43 ± 0.07 × 10⁶ and 1.42 ± 0.05 × 10⁶ Da with the concentration ranging from 0 to 60 g/l. This was the first time to discover such contribution of glycerol on γ-PGA production in Bacillus genus. And the effects of glycerol on molecular weight of γ-PGA would be developed to be an approach for the regulation of microbial γ-PGA chain length, which is of practical importance for future commercial development of this polymer.     

Estimation of the molecular weight and other parameters of macromolecules in jute by low-angle X-ray methods

Summary Low-angle X-ray scattering methods have been applied to determine the molecular volume and the molecular weight of the macromolecules present in jute fibres. The well-known low-angle scattering camera ofKratky (1) was used and the angular range investigated is from 0.17×10^–3 to 3.0×10^–3 radians. The evaluation of the parameters and the quantities were made after the theories ofGuinier (2),Kratky (3), andPorod (4).The particles in jute fibres may be of the lamella type as pointed out byKratky (30) and also recently byRoy andDas (33). The molecular volume of the macromolecules is found out to be 9.995 × 10⁶ ų . The molecular weight is 7.703×10⁶.With 5 figures and 1 table     

Novel organosoluble polynorbornene bearing a polar,pendant, ester‐bridged epoxy group via living ring‐opening metathesis polymerization

A novel organosoluble polynorbornene bearing a polar, pendant, ester‐bridged epoxy group [poly(oxiran‐2‐ylmethyl 2‐methylbicyclo[2.2.1]hept‐5‐ene‐2‐carboxylate) (polyOMMC)] was prepared via the living ring‐opening metathesis polymerization (ROMP) of active norbornenes with a Ru catalyst. PolyOMMC exhibited excellent solubility in a variety of solvents. The number‐average molecular weight of polyOMMC linearly increased with the [M]/[I] ratio (where [M] is the monomer concentration and [I] is the initiator concentration), and a narrow polydispersity of 1.09–1.19 was observed; this was considered a living polymerization. When ROMP of oxiran‐2‐ylmethyl 2‐methylbicyclo[2.2.1]hept‐5‐ene‐2‐carboxylate with [M]/[I] = 350 was carried out at 30 °C in CH₂Cl₂, the number‐average molecular weight (7.01 × 10⁴; polydispersity index = 1.07) was close to the calculated molecular weight (7.28 × 10⁴), and a diblock copolymer was observed after the addition of another monomer ([M]/[I] = 350) with an increase in the number‐average molecular weight (1.60 × 10⁵; polydispersity index = 1.11), which was close to the calculated molecular weight (1.61 × 10⁵). The modified polynorbornenes retained good solubility in methylene chloride, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, N,N‐dimethylacetamide, and N‐methyl‐2‐pyrrdione. High‐performance polynorbornenes with active epoxy groups could be designed with great potential for applications in photoresists, UV curing, and elastomers. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 4428–4434, 2006     

Unusual change in molecular weight of polyhydroxyalkanoate (PHA) during cultivation of PHA-accumulating Escherichia coli

This study aimed to investigate the factors affecting molecular weight of poly[(R)-3-hydroxybutyrate] [P(3HB)] when polyhydroxyalkanoate (PHA) synthase (PhaRC_Bsp) from Bacillus sp. INT005 was used for P(3HB) synthesis in Escherichia coli JM109. It was found that the molecular weight of P(3HB) decreased with time in mid- and late-phase of culture and was strongly affected by culture temperature. At 37 °C culture temperature, the molecular weight of P(3HB) rapidly decreased from 4.4 × 10⁵ to 4.8 × 10⁴ with culture time, whereas it was almost unchanged at 25 °C. Kinetic analysis suggested that the decrease in molecular weight of P(3HB) was due to random scission of the polymer chain. The decrease in molecular weight of P(3HB) was not observed when PHA synthases other than PhaRC_Bsp were expressed. This study sheds light on the unique behaviour in molecular weight change of P(3HB) that is synthesized by E. coli expressing PhaRC_Bsp.     

Evaluation of kinetics parameters for poly(l-lactic acid) hydrolysis under high-pressure steam

In order to evaluate more precise kinetics parameters: rate constant k and E_a values for poly(l-lactic acid) hydrolysis, the reaction was carried out under high-pressure steam in a temperature range of 100-130 °C. Molecular weights of hydrolyzates were calculated by the universal calibration method without being influenced by any weight loss. The changes in molecular weight could be successfully explained according to the auto-catalytic hydrolysis mechanism, clearly indicating the critical point. Resulting k and E_a values were estimated as 8.4 × 10⁻⁵-7.2 × 10⁻⁴ s⁻¹ and 87.2 kJ mol⁻¹ with high R² values, respectively. Moreover, to determine the deviation of the parameter values, influences of four factors on the measurements and calculation: (1) use of number-average molecular weight value alone, (2) use of relative molecular weight based on polystyrene standards, (3) weight loss during the hydrolysis, and (4) selection of reaction mechanism were evaluated quantitatively.     

Negatively charged hyperbranched polyether-based polyelectrolytes

The preparation of carboxylated hyperbranched polyglycerols of narrow polydispersity was achieved by modification (78–90%) of the hydroxyl end groups via Michael addition of acrylonitrile, followed by hydrolysis. High conversion could only be achieved for low molecular weight starting materials (520 and 1,030 g mol⁻¹). The solution properties of the resulting materials were investigated by dynamic light scattering (DLS), showing the formation of large aggregates with size depending on the pH value. After deposition on a negatively charged mica surface, the structures observed by atomic force microscope (AFM) show the coexistence of aggregates and single macromolecules. Most interesting, in the case of the lower molecular weight sample (PG 520 g mol⁻¹), extended and ordered terrace structures were formed, which are unprecedented for hyperbranched polymers and are of interest for surface modification in general.     

The Preparation of PVAc with Lower PDI Through RATRP in Miniemulsion Polymerization

In this paper, reverse atom transfer radical polymerization (RATRP) was used to prepare polyvinyl acetate (PVAc) with lower polydispersity index (PDI). The different reaction parameters such as ligand, catalyst, and surfactant were studied separately to control the polymerization of VAc. The results show that RATRP is not controlled with bpy as ligand, but it is possible to obtain PVAc with low PDI when PMDETA was used as ligand. The molecular weight and the PDI is 10.71×10⁴ and 1.62 when the molar ratio of AIBN/CuCl₂/PMDETA is 1.5:1:2, the molar ratio of AIBN to VAc is 0.5%, surfactant to deionized water is 10 wt%. The molecular weight and the PDI is 12.81×10⁴ and 1.48 when the molar ratio of AIBN/CuCl₂/PMDETA is 2:1:2. The structure of the polymer and the polymer conversion were investigated through ¹H-NMR and gravity method separately. The molecular weight (Mn) and the polydispersity of the obtained polymers were characterized through gel permeation chromatography (GPC).     

Characterization of rat liver microsomal and hepatocytal metabolites of brevetoxins by liquid chromatography–electrospray tandem mass spectrometry

Brevetoxins are natural neurotoxins that are produced by “red tide” algae. This class of compounds can cause neurotoxic shellfish poisoning and other health problems. Brevetoxin-2 is the most abundant among the nine brevetoxins that have been characterized, whereas brevetoxin-1 is the most toxic. In this study, brevetoxin-1 and brevetoxin-2 were incubated with rat liver hepatocytes and rat liver microsomes, respectively. After clean-up steps were taken to remove the proteins, samples were analyzed by liquid chromatography (LC) coupled with electrospray mass spectrometry (LC-MS). After incubation of brevetoxin-1, two metabolites were found: brevetoxin-1-M1 (molecular weight = 900 Da), and brevetoxin-1-M2 (molecular weight = 884 Da). The increase in molecular weight combined with evidence from tandem mass spectrometry showing an increased tendency for loss of water molecules, along with considerations of established precedents for chemical transformations led to the conclusion that brevetoxin-1-M1 was formed by converting one double bond in the E or F ring of brevetoxin-1 into a diol. The second metabolite (brevetoxin-1-M2) is proposed to be a hydrolysis product of brevetoxin-1 involving opening of the lactone ring with the addition of a water molecule. The incubation study of the other starting compound, brevetoxin-2, found two metabolites in the LC-ES-MS selected ion chromatogram. Brevetoxin-2-M1 (molecular weight = 912 Da) gave a large [M−H]⁻ peak at m/z 911, and its product ion mass spectrum allowed the deduction that this metabolite was the hydrolysis product of brevetoxin-2 involving conversion of the lactone to a carboxylic acid and an alcohol. The second metabolite (brevetoxin-2-M2, molecular weight = 896 Da) was deduced to have the same structure as that of brevetoxin-3 based on identical chromatographic retention times and similar mass spectra as those obtained for a brevetoxin-3 standard.     

Fractionation and physico-chemical analysis of degraded lignins from the black liquor of Eucalyptus pellita KP-AQ pulping

The degraded Eucalyptus pellita kraft lignin from the black liquor of KP-AQ pulping was precipitated directly at pH ∼2.0 without further purifying, since the lignin obtained is more representative with a whole distribution of molecular weight. The precipitated lignin was fractionated into six fractions by successive extraction with organic solvents. A comparison study of the lignin heterogeneity between the fractions was made in terms of fractional yield, content of associated polysaccharides, alkaline nitrobenzene oxidation, molecular weight distribution, ¹H NMR and ¹³C NMR spectroscopy and thermal stability. It was found that the lignin fractions contained higher associated hemicelluloses and ratios of non-condensed syringyl/guaiacyl units which were extracted by organic solvents with higher Hildebrand solubility parameters. The results from GPC and TGA showed that the polydispersity and the thermal stability of the lignin fractions increased with increasing molecular weight. In the low molecular weight fraction, small amounts of β-aryl ether bond (β-O-4) surviving the KP-AQ pulping were detected by both ¹H and ¹³C NMR spectra.     

Molecular characterization of branched polysaccharides from Tremella fuciformis by asymmetrical flow field‐flow fractionation and size exclusion chromatography

To accurately characterize branched polysaccharides with high molecular weights from medicinal and edible mushrooms and identify the limitations of size exclusion chromatography, molecular characteristics of polysaccharides from Tremella fuciformis were determined and compared by asymmetrical flow field‐flow fractionation coupled with multiangle laser light scattering and refractive index detection, and size exclusion chromatography coupled with multiangle laser light scattering and refractive index detection, respectively. Results showed that molecular weights of three batches of T. fuciformis polysaccharides were determined as 2.167 × 10⁶ (TF1), 2.334 × 10⁶ (TF2), and 2.435 × 10⁶ Da (TF3) by size exclusion chromatography, and 3.432 × 10⁶ (TF1), 3.739 × 10⁶ (TF2), and 3.742 × 10⁶ Da (TF3) by asymmetrical flow field‐flow fractionation, as well as 3.469 × 10⁶ Da (TF1) by off‐line multiangle laser light scattering, respectively. Results suggested that size exclusion chromatography was unable to accurately characterize T. fuciformis polysaccharides, which may be due to its limitations such as shear degradation and abnormal coelution. Compared to size exclusion chromatography, asymmetrical flow field‐flow fractionation could be a better technique for the molecular characterization of branched polysaccharides with high molecular weights from medicinal and edible mushrooms, as well as from other natural resources.