Study of chain branching in natural rubber using size-exclusion chromatography coupled with a multi-angle light scattering detector (SEC-MALS)

The different rheological behaviour of natural rubber (NR) compared to industrial synthetic poly(cis-1,4-isoprene) (SR) has been attributed to the gel phase and long-chain branching. Previous studies on branching in NR were carried out using the fractionation technique by precipitation to obtain narrow molar mass distribution. In this study, chain branching of poly(cis-1,4-isoprene) in NR was characterised by size-exclusion chromatography coupled with an online multi-angle light scattering detector (SEC-MALS). The nanoaggregates adsorbed on the column packing interfered with branching characterisation for short and medium chains (M_w < 1000 kg/mol). Using a master curve of linear standard poly(cis-1,4-isoprenes), SEC-MALS revealed no or very little branching in the higher chains (1000 < M_w < 10,000 kg/mol) of natural rubber contrary to previous studies. This study showed that the soluble portion of NR samples was composed of almost linear poly(cis-1,4-isoprene) and nanoaggregates with rather compact structures.     

Molecular weight determination of hypromellose phthalate (HPMCP) using size exclusion chromatography with a multi-angle laser light scattering detector

The molecular weight of hypromellose phthalate (HPMCP), a polymer used for enteric coating, was determined using size exclusion chromatography with a multi-angle laser light scattering detector. The values of weight-average molecular weight (Mw) of commercially available grades (HP-55, HP-55S, and HP-50) were 45600, 60200, and 37900, respectively. Their inter-day precisions expressed in terms of the coefficient of variation were less than 3%. A correlation curve between Mw and solution viscosity was prepared so that Mw could be easily estimated from the solution viscosity measured by the compendial method.     

Molecular weight determination of hypromellose acetate succinate (HPMCAS) using size exclusion chromatography with a multi-angle laser light scattering detector

The molecular weight of hypromellose acetate succinate (HPMCAS), a polymer used for enteric coating, was determined by means of size exclusion chromatography with a multi-angle laser light scattering detector. The weight-average molecular weight (Mw) of several lots and grades ranged approximately from 17000 to 20000, and the number-average molecular weight (Mn) was around 13000. The inter-day precision of measurement, in terms of the coefficient of variation, was less than 5%.     

Molar mass characterization of cationic methyl methacrylate-ethyl acrylate copolymers using size-exclusion chromatography with online multi-angle light scattering and refractometric detection

Size-exclusion chromatography (SEC) combined with online multi-angle light scattering (MALS) and refractometric (RI) detection has been employed for the molar mass characterisation of water-insoluble cationic methyl methacrylate-ethyl acrylate copolymers (Eudragit RS and RL). Due to their positive charge, cationic polymers are particularly difficult to separate on a SEC column, in worst cases being completely adsorbed on the oppositely charged packing material. This work has examined how a careful addition of salt (LiCl) to the copolymer solution in ethanol decreases the electrostatic interactions, clearly seen as a decrease in elution volume from the SEC column as well as an improved recovery. At a certain level of ionic strength, typically about 50 mM, the copolymer recovery from the SEC column reached 100% and molar mass distributions corresponding to the complete sample could be obtained. The combined MALS/RI detection gives the opportunity to measure the absolute molar mass independent of recovery and retention. Thus, in this study, it turned out to be a favourable tool for tracing the changes in elution behaviour of the charged copolymer as the ionic strength was increased.     

Accurate determination of the length of carbon nanotubes using multi-angle light scattering

We have prepared a kind of molecularly imprinted nano-porous sensing film for the adsorption of melamine. It consists of a graphite electrode impregnated with paraffin and modified with melamine, chitosan, silver nanoparticles and polyquercetin by employing an electrochemical method. The film displays excellent and highly selective sorption of melamine in the 3-dimensional porous nanomaterial, and this was applied to the determination of melamine in dairy products. The electrode responds linearly to melamine in the concentration range of 1?×?10^?8 to 9?×?10^?7?M, with a detection limit of 1.3?×?10^?9?M (at 3??) in real samples, and with recoveries in the range of 99 to 102%. The surface structure and composition of the sensor was characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, infrared spectroscopy, and electrochemical techniques. The interaction between the porous film and melamine was also studied by using hexacyanoferrate (III) as an electrochemical indicator.

Characterization of lignin using multi-angle laser light scattering and atomic force microscopy

Small differences in the isolation techniques of lignin can result in significant changes in its molecular structure and configuration. Light scattering (evaluated at 18 different angles in a plane), Atomic Force Microscopy (AFM) and Near Infrared Spectroscopy (NIR) proved very effective for evaluating the characteristics of lignin. Zimm plots were generated using Zimm, Debye and Berry formalisms to evaluate the weight average molecular weight (MW), radius of gyration (r_g), hydrodynamic radius (r_h) and second virial coefficient (A₂). Two types of lignin and nine different solvents were used for the study, to analyze the conformation of lignin molecules in different solvents expected to be used in lignin degradation and subsequent analysis. Absolute MW and r_g decreased and the dn/dc increased when the solvent used for lignin was changed from water to sodium hydroxide. The two types of lignin also exhibited different values for all the above estimated parameters. This study also highlighted the differences between the unlyophilized and lyophilized lignin in terms of aggregation, pH dependence and stability over time. This aggregation has never been seen on a ultraviolet (UV) or refractive index (RI) detector that has been used so far for liquid chromatography (LC) reducing the reliability of lignin depolymerization data obtained without light scattering.     

Equilibration of mixtures of polyamic acids studied using size-exclusion chromatography and light scattering

The transamidation reaction in a polyamic acid solution has been investigated using size-exclusion chromatography and low-angle light scattering. Mixtures of a high-molecular weight (DP = 150) and a low-molecular weight (DP = 10) polymer and of the high-molecular weight polymer with monomer were studied. Mixtures were made at high and low concentrations. The polyamic acid studied is the product of the polycondensation of 3,3′,4,4′-biphenyltetracarboxylic acid dianhydride (BPDA) with oxydianiline (ODA). In all cases the molecular weight distribution equilibrated with time to a most-probable distribution with a DP consistent with the stoichiometry of the mixture. Equilibration required about 2 weeks for mixtures of 10% by weight at ambient temperatures. The effect of addition of a small amount (5%) of low-molecular weight material to sample of high-molecular weight is dramatic; for DP = 150 the molecular weight is decreased by more than one-half. In an entangled solution or melt, this would reduce the viscosity by an order of magnitude.     

Measurement of uncertainty in peptide molecular weight determination using size-exclusion chromatography with multi-angle laser light-scattering detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The molecular weight of biopolymers such as peptides or proteins is vital information for understanding their physical/chemical properties. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and size-exclusion chromotography-multi-angle laser light scattering (SEC-MALLS) techniques each with its advantages and limitations were used for molecular weight determination of the SPf66 peptide. The precision of each method was studied using a two-factor fully-nested design with all the analyses performed by the same operator on a single instrument. The overall precision corresponded to the time-different intermediate precision (i.e. time and repeatability conditions). In the case of SEC-MALLS, all potential uncertainty components were carefully evaluated in an Ishikawa diagram, then included and mathematically combined with the uncertainty arising from the accuracy assessment to provide the overall uncertainty. In this case, the refractive-index increment with solute concentration value (dn/dc) provided the most significant contribution to the combined uncertainty. A method for its quantitation is proposed. The accuracy of method B (SEC-MALLS) against reference method A (MALDI-TOF-MS) was assessed using the interval testing hypothesis to limit the risk of unacceptable bias. The results indicate that the bias of B is higher than the limit established at 5%, and is therefore not traceable to A under the studied conditions.     

Size fractionation and characterization of natural colloids by flow-field flow fractionation coupled to multi-angle laser light scattering

Flow-field flow fractionation (FlFFF) coupled to multi-angle laser light scattering (MALLS) was evaluated for size and shape determination of standard spherical and arbitrarily shaped natural colloids. Different fitting methods for light scattering data retrieved from MALLS were evaluated to determine the particle size of spherical standards and natural colloids. In addition, FlFFF was optimized for best fractionation in connection to MALLS, minimal colloids-membrane interaction, and minimal sample losses. FlFFF, calibrated with standard particles, was used to determine hydrodynamic diameter, or radius (D(h) or R(h)), of the fractionated colloids, whereas the MALLS was used to determine root mean square radius of gyration (R(g)) for fractionated colloids. Combining both results, by calculating the R(g)/R(h) ratio, allows an estimation of colloid deviation from the shape of homogeneous sphere. Accordingly, this study demonstrates that, FlFFF-MALLS is a valuable technique for characterizing heterogeneous and arbitrarily shaped natural colloidal particles in terms of size and shape. To check the usefulness of FlFFF-MALLS in natural colloid studies, the technique was used to investigate the sedimentation behavior of extracted soil colloidal particles. Results illustrate that, in a silty till sample, carbonates function as cement between the colloidal particles, and consequently, change their sedimentation behavior. On the other hand, carbonate dissolution generates a more homogeneous colloidal sample.     

Evaluation of on-line high-performance size-exclusion chromatography, differential refractometry, and multi-angle laser light scattering analysis for the monitoring of the oligomeric state of human immunodeficiency virus vaccine protein antigen

Chiron has developed a novel mutant form of the human immunodeficiency virus (HIV) envelop protein, o-gp140, that is currently entering Human Phase 1 clinical trials for testing as a prophylactic HIV vaccine. The o-gp140 protein is oligomeric and the quaternary structure is thought to play an important role in its activity as an antigen. As o-gpl40 proceeds through the clinical trial process and prior to marketing approval, analytical methods that are able to demonstrate manufacturing consistency with respect to degree of oligomerization will need to be developed and validated. On-line high-performance size-exclusion chromatography, differential refractometry, and multi-angle laser light scattering analysis (HPSEC-RI-MALLS), a method commonly used to obtain the molar mass of macromolecules based on the Rayleigh-Gans-Debye approximation, was evaluated for this purpose. The results obtained demonstrated intra- and inter-day precisions to be 0.9 and 3.6% R.S.D., respectively. Accuracy was found to be equal to, or better than, 11% when comparing the known molar masses of test proteins to that of the molar masses determined by the method. Additionally, the method compared favorably to orthogonal native polyacrylamide gel electrophoresis and ultracentrifugation analyses. R-factor analysis was used to demonstrate that HPSEC-RI-MALLS is capable of discriminating compositional differences between o-gpl40 test lots. Based on the data presented, HPSEC-RI-MALLS may be a suitable manufacturing control method.     

Analysis of aggregates of human immunoglobulin G using size-exclusion chromatography,static and dynamic light scattering

Large aggregates (Mr: 10(6)-10(7) g/mol) of human immunoglobulins are present in extremely small concentrations in IgG preparations (<0.1%). Traces of large protein aggregates cannot be determined by conventional size-exclusion chromatography (SEC) using UV detection due to limitations in sensitivity. The conventional analysis of IgG by SEC is limited to dimers and oligomers. Using light scattering it is possible to determine significant differences concerning the aggregate composition and the extent of protein aggregation in samples of different process steps. Two different pilot preparations were analyzed by SEC with UV and static light scattering detection and compared to dynamic light scattering in the batch mode. The change of large aggregates could be monitored and data were corroborated by dynamic light scattering.     

Calculation of the binary diffusion second virial coefficient

Calculations of the first density correction to the binary diffusion coefficient are presented for several mixtures. These calculations are based on the classical kinetic theory for mixtures developed by Bennett and Curtiss. The theoretical predictions agree well with experimental data.     

Intermolecular potential and second virial coefficient of the water-hydrogen complex

We construct a rigid-body (five-dimensional) potential-energy surface for the water-hydrogen complex using scaled perturbation theory (SPT). An analytic fit of this surface is obtained, and, using this, two minima are found. The global minimum has C2v symmetry, with the hydrogen molecule acting as a proton donor to the oxygen atom on water. A local minimum with Cs symmetry has the hydrogen molecule acting as a proton acceptor to one of the hydrogen atoms on water, where the OH bond and H2 are in a T-shaped configuration. The SPT global minimum is bound by 1097 microEh (Eh approximately 4.359744 x 10(-18) J). Our best estimate of the binding energy, from a complete basis set extrapolation of coupled-cluster calculations, is 1076.1 microEh. The fitted surface is used to calculate the second cross virial coefficient over a wide temperature range (100-3000 K). Three complementary methods are used to quantify quantum statistical mechanical effects that become significant at low temperatures. We compare our results with experimental data, which are available over a smaller temperature range (230-700 K). Generally good agreement is found, but the experimental data are subject to larger uncertainties.     

Intermolecular potential and second virial coefficient of the water-nitrogen complex

The authors construct a rigid-body (five-dimensional) potential energy surface for the water-nitrogen complex using the systematic intermolecular potential extrapolation routine. The intermolecular potential is then extrapolated to the limit of a complete basis set. An analytic fit of this surface is obtained, and, using this, the global minimum energy is found. The minimum is located in an arrangement in which N2 is near the H atom of H2O, almost collinear with the OH bond. The best estimate of the binding energy is 441 cm-1 (1 cm-1 approximately 1.986 43x10(-23) J). The extrapolated potential is then used to calculate the second cross virial coefficient over a wide temperature range (100-3000 K). These calculated second virial coefficients are generally consistent with experimental data, but for the most part the former have smaller uncertainties.     

Influence of polymolecularity on the second virial coefficient of polymers

Scaling theory is applied to derive expressions describing the influence of polymolecularity on the second virial coefficient, A₂, as obtained from osmotic pressure and light scattering measurements. Numerical values of polymolecularity correction factors are calculated for Schulz-Zimm and logarithmic normal distributions of the molecular weight, different qualities of the solvent and several ratios of the weight-average and the number-average molecular weights M̄_w/M̄_n. It is found that in the equation $ A_2 = K_{A_2 } \cdot M_{{\rm av}}^{a_{A_2 } } $ the weight-average molecular weight is a good approximation for M_av if A₂ is measured via light scattering, while the number-average molecular weight can be inserted for M_av if A₂ stems from osmotic pressure measurements.     

Determination of lignin by size exclusion chromatography using multi angle laser light scattering

A method was developed using high-performance size exclusion liquid chromatography (HPSEC) with multi-angle laser light scattering (MALLS), quasi-elastic light scattering (QELS), interferometric refractometry (RI) and UV detection to characterize and monitor lignin. The combination proved very effective at tracking changes in molecular conformation of lignin molecules over time; i.e. changes in molecular weight distribution, radius of gyration, and hydrodynamic radius. Until this study, UV detection (280 nm) had been the primary lignin determination method for chromatography. Three different HPLC columns were used to study the effects of pH, flow conditions, and mobile phase compositions (dimethyl sulphoxide, water, 0.1M NaOH, and lithium bromide) on the chromatography of lignin. Since light scattering accuracy is highly dependent on solute concentration, both the UV and RI detectors were calibrated for use as concentration detectors. Shodex Asahipak GS-320 HQ column with 0.1M NaOH (pH 12.0) run at 0.5 ml/min was found to give the highest separation and most consistent recovery. The study also revealed that the lignin aggregated at pH below 8.5. This aggregation was detected only by MALLS and was not observed on UV or RI detectors. It is very important to take this loss in apparent concentration due to aggregation into consideration before collecting reliable depolymerization data.     

Behavior of polysaccharide assemblies in field-flow fractionation and size-exclusion chromatography

Asymmetric flow field-flow fractionation (AsFlFFF) and high-performance size-exclusion chromatography (HPSEC) are techniques for separating and characterizing macromolecules; until now the latter is more utilized for analyzing polysaccharides. The demand for characterizing complex, high-molar-mass polysaccharides has raised interest in the use of AsFlFFF in analyzing polymeric carbohydrates in addition to HPSEC. In this paper, we compare the behavior of arabinoxylan aggregates present in aqueous solution in AsFlFFF and HPSEC and their effect on the obtained molecular characteristics (molar mass averages and size). Although the amount of aggregates in aqueous arabinoxylan solutions may be low, their role needs to be understood to avoid erroneous interpretations of AsFlFFF and HPSEC data. When these two separation systems were compared, AsFlFFF seemed to possess more separation power for the differentiation of aggregates from individual chains than HPSEC. To our knowledge, this is the first report on the characterization of xylans with AsFlFFF.     

Utilization of an evaporative light scattering detector for high-performance size-exclusion chromatography of galacturonic acid oligomers

A high-performance size-exclusion chromatography-evaporative light scattering detector method was used to separate, detect and quantify galacturonic acid (GA) oligomers. In 40 mM acetic acid GA monomer, dimer and trimer could be separated with baseline resolution but polygalacturonic acid (PGA) precipitated and could not be eluted from the column. An NH4OAc, pH 3.7, buffer was developed as the eluent which separated GA oligomers as well as PGA and pectin without precipitation. Linear calibration curves for mono-, di- and tri-GA were produced with this buffer which could be used to estimate masses of tetra-, penta- and hexa-GA, as well as 19mer and 20mer.