SEC Separation Research Articles

Characterization of Unsaturated Polymers Using High‐Temperature Size Exclusion Chromatography Coupled with Ultraviolet‐Evaporative Light Scattering Dual Detection

AbstractSize exclusion chromatography (SEC) is a technique routinely used at high temperature to analyze the molar mass distribution (MMD) in polymers. The distribution of comonomer along the molar mass axis in a copolymer is a key microstructural feature that determines the macroscopic properties of the material and thus its range of possible applications. However, unsaturated polymers requiring high dissolution temperature, measurement of chemical composition distribution (CCD) along the molar mass axis, have so far been hampered by the lack of suitable detection methods. Toward this goal, the potential of ultraviolet (UV) detection for high temperature SEC of ethylene‐styrene, propylene‐styrene copolymers, and ethylene‐propylene‐diene terpolymers is investigated. The influence of experimental parameters such as analyte concentration, unsaturation content, and mobile phase on the spectroscopic response is probed. An evaporative light scattering detector (ELSD) is employed to monitor the analyte concentration, in combination with the UV detector, to quantitatively measure the distribution of unsaturation content along the MMD. A slice‐by‐slice UV/ELSD ratio is calculated to determine the average chemical composition of each molar mass fraction after SEC separation.

ITRAQ-based quantitative proteomic analysis of key barley proteins reveals changes after malting

Since the choice of malting varieties, as well as the impact of malting on protein composition and other malt quality parameters, are fundamental for beer production, there was a need to focus on this study. The present work was undertaken to evaluate the effect of malting processing on the barley proteins. The relationship between the protein pattern of barley grains and malt was investigated by combination of HPLC SEC separation with MS. We have found that the biggest difference between grain and malt protein profiles was observed in the fraction, where these proteins were identified: β-amylase, β-glucosidase and two various isoforms of protein Z (Z4, Z7). This peak was significantly increased after the malting. In addition, iTRAQ was applied for investigation of the relative quantitative changes of selected proteins after food technological processing. Our results indicate that the content of β-amylase is increased more rapidly in comparison to β-glucosidase. On the other hand, protein Z7 seems to have the opposite trend. The quantitative analysis reveals the increasing tendency of protein Z4 and decreasing tendency of Z7 protein after malting.

SEC Separation of Polysaccharides Using Macroporous Spherical Silica Gel as a Stationary Phase

Meso- and macroporous spherical silica gels of pore sizes in the range of 60–1000 Å and 40–75 µm particle size were investigated as a stationary phase for the separation and purification of polysaccharides and poly(ethylene glycols) (PEGs) of various MWs using an aqueous mobile phase. Sephadex and Bio-Gel were used for comparison as the most common stationary phases for similar purposes. The separation of dextrans of a mean MW = 31 kDa from small molecules (NaCl) was possible with SiO2 with a pore size of 60–300 Å, but the observed efficiencies of a column of the same size were lower comparing with Sephadex or Bio-Gel. In the case of oxidized alginic acid only SiO2 of the 60 Å pore size was suitable, while Sephadex, Bio-Gel and other investigated silicas were not efficient. Sephadex and 300–1000 Å SiO2 offered the possibility of dividing dextrans with MW within the range of 1 MDa–10 kDa into fractions of various MWs, while Bio-Gel and 60 Å SiO2 were not suitable. The investigated silica gels strongly adsorbed PEGs of MW 2–20 kDa. The amount adsorbed decreased with the increase of pore size and they were not useful as a stationary phase for this class of polymers. An advantage of SiO2 of the investigated particle size was a very low back pressure comparing with Sephadex. A considerably lower price of silica offers time- and cost-efficient separation of polysaccharides.Graphical

Simultaneous size characterization and mass quantification of the in vivo core-biocorona structure and dissolved species of silver nanoparticles

Size characterization of silver nanoparticles with biomolecule corona (AgNP@BCs) and mass quantification of various silver species in organisms are essential for understanding the in vivo transformation of AgNPs. Herein, we report a versatile method that allows simultaneous determination of the size of AgNP@BCs and mass concentration of various silver species in rat liver. Both particulate and ionic silver were extracted in their original forms from the organs by alkaline digestion, and analyzed by size exclusion chromatography combined with inductively coupled plasma mass spectrometry (SEC-ICP-MS). While the silver mass concentrations were quantified by ICP-MS with a detection limit of 0.1μg/g, the effective diameter of AgNP@BCs was determined based on the retention time in SEC separation with size discrimination of 0.6–3.3nm. More importantly, we found that the BC thickness of AgNP@BCs is core size independent, and a linear correlation was found between the effective diameter and core diameter of AgNP@BCs in extracted tissues, which was used to calibrate the core diameter with standard deviations in the range of 0.2–1.1nm. The utility of this strategy was demonstrated through application to rat livers in vivo. Our method is powerful for investigating the transformation mechanism of AgNPs in vivo.

Size Exclusion Chromatography-Ion Mobility-Mass Spectrometry Coupling: a Step Toward Structural Biology.

Noncovalent interactions are essential for the structural organization of biomacromolecules in cells. For this reason, the study of the biophysical, dynamic, and architectural interactions among biomacromolecules is essential. Since mass spectrometry requires compatible solutions while preserving the noncovalent bonding network, we envisioned that size exclusion chromatography coupled with ion mobility and mass spectrometry would be a valuable technique to desalt the initial sample and provide solution and gas-phase structural information in a single stage experiment. Such coupling allowed obtaining information on solution protein complex composition with SEC separation and on authenticity and purity with IMS-MS. Our study demonstrated that such coupling is compatible, useful, as well as suitable for a routine analysis, in pharmaceutical industry, for example. Mobility data were reliable and injected standards allowed calibrating the collision cross-section scale. Graphical Abstract ᅟ.

Simulating a dual beam combiner at SUSI for narrow-angle astrometry

The Sydney University Stellar Interferometer (SUSI) has two beam combiners, i.e. the Precision Astronomical Visible Observations (PAVO) and the Microarcsecond University of Sydney Companion Astrometry (MUSCA). The primary beam combiner, PAVO, can be operated independently and is typically used to measure properties of binary stars of less than 50 milliarc- sec (mas) separation and the angular diameters of single stars. On the other hand, MUSCA was recently installed and must be used in tandem with the for- mer. It is dedicated for microarcsecond precision narrow-angle astrometry of close binary stars. The performance evaluation and development of the data reduction pipeline for the new setup was assisted by an in-house computer simulation tool developed for this and related purposes. This paper describes the framework of the simulation tool, simulations carried out to evaluate the performance of each beam combiner and the expected astrometric precision of the dual beam combiner setup, both at SUSI and possible future sites.

Determining the Mark–Houwink parameters of nitrile rubber: a chromatographic investigation of the NBR microstructure

The microstructure of acrylonitrile–butadiene rubber (NBR) was shown to be dependent on the polymerization conditions. The NBR investigated in the current study was obtained via radical copolymerization under azeotropic conditions (AN/BD = 38/62) in organic solution in the presence of either a conventional chain transfer agent or a reversible addition fragmentation chain transfer (RAFT) agent. The variation in the polymer microstructure was proven to originate from different radical environments during the polymerizations with initial radical initiator concentrations in the polymerizations studied ranging from 1.0 mM to 34.1 mM. The variation of the polymer microstructure was evidenced by triple SEC measurements, making use of the simultaneous determination of molecular weights with two independent methods, namely on-line viscometry and on-line light scattering. It is additionally evidenced that the microstructure shows a gradual variation during the course of a polymerization, a behaviour observed when the polymerizations were performed in the presence of an elevated initial radical initiator concentration. Furthermore, experimental evidence for the variation of the NBR microstructure during RAFT polymerizations is provided. At low conversions, a rather uniform polymer is obtained. With increasing conversion, a loss of the controlled character is observed and the microstructures approach those of nitrile rubber obtained by conventionally controlled free radical copolymerizations employing mercaptane transfer agents. Despite the differences in the polymer microstructure, it is possible to report a single set of MHKS parameters for the prepared NBR with azeotropic composition. A linear regression of the Mark–Houwink plots of the samples polymerized under different conditions gives values of K = (49.5 ± 5.5) × 10−5 dL g−1 and α = 0.689 ± 0.010 with a low error margin for SEC separation in THF at 25 °C. Weight average molecular weights of the investigated NBR samples were in the range of 40000 to 155000 g mol−1. The molecular weights of the copolymers determined via universal calibration with the MHKS parameters presented in the current study show a good agreement with molecular weights obtained from light scattering, underpinning the veracity of the obtained parameters.

Size-exclusion-chromatography separation of randomly branched polymers with tetrafunctional branch points and local dispersity

The SEC separation of a randomly branched polymer, in particular local dispersity due to branching, are theoretically examined. A model of the SEC separation of randomly branched polymer with tetrafunctional branch points enabling the estimation of local dispersity was developed. Measurable quantities (branching indices) were compared with real data. Local dispersity due to branching is demonstrated to depend on elution volume and degree of branching and in the area of the beginning of the elution curve it can reach non-negligible values.

SEC‐MR‐NMR: Online Coupling of Size Exclusion Chromatography and Medium Resolution NMR Spectroscopy

Online coupling of size exclusion chromatography together with medium resolution nuclear magnetic resonance (SEC-MR-NMR) might be one solution to the problem of chemically sensitive detection in liquid polymer chromatography. By use of a combination of SEC with a table-top, specially designed 20 MHz NMR spectrometer, based on a permanent magnet, online (1) H NMR spectra of SEC fractions can be obtained. The integration of digital filters, mechanical shims and electronic shims led to substantially improved sensitivity and chemical selectivity compared to former TD (time domain) 20 MHz instruments. (1) H NMR spectra of PMMA and PS homopolymers as well as PS-PMMA block copolymers were of sufficient quality to enable detection and de-formulation of unknown polymer compounds. (1) H NMR spectra of acceptable resolution and S/N ratio were collected online during the chromatography. The SEC separation online with the NMR measurements performed well and resulted in the proof of principle of the SEC-MR-NMR combination.

High-temperature two-dimensional liquid chromatography of ethylene-vinylacetate copolymers

Temperature rising elution fractionation hyphenated to size exclusion chromatography (TREF × SEC) is a routine technique to determine the chemical heterogeneity of semicrystalline olefin copolymers. Its applicability is limited to well crystallizing samples. High-temperature two-dimensional liquid chromatography, HT 2D-LC, where the chromatographic separation by HPLC is hyphenated to SEC (HPLC × SEC) holds the promise to separate such materials irrespective of their crystallizability. A model blend consisting of ethylene-vinyl acetate (EVA) copolymers covering a broad range of chemical composition distribution including amorphous and semicrystalline copolymers and a polyethylene standard was separated by HT 2D-LC at 140 °C. Both axes of the contour plot, i.e. the compositional axis from the HPLC and the molar mass axis from the SEC separation were calibrated for the first time. Therefore, a new approach to determine the void and dwell volume of the developed HT 2D-LC instrument was applied. The results from the HT 2D-LC separation are compared to those from a cross-fractionation (TREF × SEC) experiment.

Size exclusion chromatography coupled to electrospray ionization mass spectrometry for analysis and quantitative characterization of arsenic interactions with peptides and proteins

Arsenic-binding proteins are of toxicological importance since enzymatic activities can be blocked by arsenic interactions. In the present work, a novel methodology based on size exclusion chromatography coupled to electrospray ionization mass spectrometry (SEC-ESI-MS) was developed with special emphasis to preserve the intact proteins and their arsenic bindings. The eluent composition of 25 mMTris/HCl, pH 7.5, with the addition of 100-mM NaCl optimized for SEC with UV detection provided the highest SEC separation efficiency, but was not compatible with the ESI-MS because of the non-volatility of the buffer substance and of the salt additive. In order to find the best compromise between chromatographic separation and ionization of the arsenic-binding proteins, buffer type and concentration, pH value, portion of organic solvent in the SEC eluent as well as the flow rate were varied. In the optimized procedure five different arsenic-binding peptides and proteins (glutathione, oxytocin, aprotinin, alpha-lactalbumin, thioredoxin) covering a molar mass range of 0.3-14 kDa could be analyzed using 75% 10-mM ammonium formate, pH 5.0/25% acetonitrile (v : v) as eluent and a turbo ion spray source operated at 300 degrees C and 5.5 kV. A complete differentiation of all peptides and proteins involved in the arsenic-binding studies as well as of their arsenic-bound forms has become feasible by means of the extracted ion chromatograms (XIC) of the mass spectrometric detection. The new method offered the possibility to estimate equilibrium constants for the reaction of phenylarsine oxide with different thiol-containing biomolecules by means of the XIC peak areas of reactants and products. Limits of detection in the range of 2-10 microM were obtained by SEC-ESI-MS for the individual proteins.

Studying metal integration in native and recombinant copper proteins by hyphenated ICP-DRC-MS and ESI-TOF-MS capabilities and limitations of the complementary techniques

The complementary use of LC-ESI-MS and LC-ICP-MS for characterization of native and recombinant copper proteins (molar mass range 10–20 kDa) was investigated. SEC and IC separation protocols were implemented for hyphenated ICP-MS analysis. The studies showed that validation of the methods addressing metal integration on a quantitative basis via metal to sulfur ratios demanded complementary determinations of molar mass. Reversed phase LC-ESI-TOF-MS measurements showed point mutation for the investigated recombinant apo-plastocyanin. Moreover, both recombinant proteins, i.e. plastocyanin and the CuA domain of cytochrome c oxidase from the cyanobacterium Synechocystis, lost their N-terminal amino acid upon expression. Since in both cases methionine formed the N-terminus the theoretical metal to sulfur ratio of the protein was changed. Excellent precision ranging at 3 ppm (N = 5) could be achieved for the determination of multiply charged ion patterns by LC-ESI-TOF-MS. The precision of the molar mass determination after deconvolution ranged at 5–15 ppm (N = 5). The intact metal containing copper proteins were measured by flow injection-ESI-TOF-MS under non-denaturing conditions (pH 5). Mass accuracy of ESI-TOF-MS allowed confirming not only stoichiometry of metal ligation but also the oxidation state of the metal center in plastocyanin and the CuA domain of cytochrome c oxidase. Moreover, IC-ICP-MS measurements on isotopically enriched Cu proteins were accomplished showing the potential of hyphenated ICP-MS analysis in future tracer studies.

Data processing method for the determination of accurate molecular weight distribution of polymers by SEC/MALDI-MS.

A novel data processing method for a hyphenated technique, size exclusion chromatography/matrix-assisted laser desorption/ionization-mass spectrometry (SEC/MALDI-MS), has been proposed to determine accurate molecular weight distributions on the basis of the individual oligomer species of a polymer. This method is based on the concept that the individual peak intensities of MALDI mass spectrum observed for every SEC fraction with narrow molecular weight distribution could be adjusted to the quantified values to reveal the accurate molecular weight distribution using the signal intensity of the corresponding fraction on the SEC chromatogram observed with a refractive index detector. At first, the theory of the proposed date processing is described in detail. Then, experimental verification of the method is described. This was performed through the characterization of mixtures of three kinds of monodispersed polystyrene reference materials (weight average molecular weight = ca. 6000, 10000, and 18000) as model samples. An accurate trimodal molecular weight distribution for the individual oligomer species of the sample was obtained without any influence of the chromatographic band broadening observed in the original SEC chromatogram. Moreover, the method for depicting the elution profiles of individual oligomer species during SEC separation was also obtained as a "mass chromatogram" using the data processing procedure.

Fractal calibration in size-exclusion chromatography: I. An introduction

The elution behaviour of different polymer–solvent systems in three types of organic columns for SEC has been compared and interpreted. The experimental data show that the classical universal calibration is not accomplished. Deviations from a unique curve are observed due to the binary and ternary interactions between the components of the system (solvent, polymer and gel) which results on secondary mechanisms accompanying the main pure or “ideal” SEC separation mechanism. Both, enthalpic and entropic effects are interpreted in terms of the swelling and crosslinking degrees of the gel packings, and are also related with the fractal characteristics of their surfaces, such as the fractal dimension and the available pore size. Moreover, a relationship between the fractal dimension of the pore surface and the chromatographic distribution coefficient is proposed.

Effect of metal ions on the molecular weight distribution of humic substances derived from municipal compost: ultrafiltration and size exclusion chromatography with spectrophotometric and inductively coupled plasma-MS detection.

The effect of metal ions (Co, Cu, Ni, Pb, Zn) on the molecular weight distribution of humic substances (HSs) obtained from compost is studied. We believe this is the first of this type of study applied in this way to humic substances. Size exclusion chromatography is coupled with two on-line detection systems (spectrophotometric and ICPMS) to study the binding of metal ions by humic substances leached from compost. ICPMS provided highly specific, sensitive, and multielement analytical information that enabled obtaining direct experimental evidence for the participation of metal ions in molecular size distributions of humic compounds. The compost extract or its high molecular weight fraction (>5,000) was put in contact with EDTA or citrate ions, thereby competing with HSs for binding metals. The experiments were carried out by varying the pH maintained by Tris-HCl or CAPS buffer (pH 8.0 and 10.3) and keeping the ionic strength constant. The elution profile of humic substances using UV/ visible detection was compared with those from ICPMS detection of Co, Cu, Ni, Pb, and Zn in the same chromatographic runs. The results obtained suggested that both bridging between small molecules and complexation/ chelation by individual molecules are involved in metal ion binding to humic substances. The use of ICPMS to study the role of metal ions in aggregation/disassociation of humic substances proposed in this work is promising. Coupling element-specific detection with SEC or other separation systems allows better understanding of the mobility and bioaccessibility of elemental species in the environment and further elucidation of the dissolved humic structure.

On-line size-exclusion chromatography/electrospray ionisation mass spectrometry of aquatic humic and fulvic acids

Isolated aquatic humic and fulvic acids were analysed with on-line size exclusion chromatography/electrospray ionisation mass spectrometry (SEC/ESI-MS). An eluent composition which enabled electrospray ionisation was identified. The SEC separation improved interpretability of mass spectra and may open up new possibilities for molecular weight determination of humic substances. A linear dose-response relationship over a factor of 20 was obtained and the limit of detection was 50ng/uL for humic and fulvic acids. Spectral changes due to different ionisation conditions (pH and cone voltage) were investigated. A natural water sample from a Swedish lake was analysed. Copyright 2000 John Wiley & Sons, Ltd.

Degradation of High Molecular Weight Polystyrenes During the SEC Separation Process, as Demonstrated by SEC Coupled with Lalls and by Static Light Scattering

In order to investigate the phenomenon of polymer degradation during the SEC separation process, we compared the weight-average molecular weights of a series of polystyrene (PS) samples with Mw going from 2.3×103 to 1.84×107, which were determined by static light scattering (LS) and by SEC-LALLS using various crosslinked PS gels. For all the gels considered, there was a good agreement between LS and SEC-LALLS values for Mw up to 2×106. For higher Mw, the values given by SEC-LALLS were lower than those given by LS; the decrease in Mw was different in various SEC columns. Since the same chromatographic equipment was used for all the SEC-LALLS measurements, we presumed that the decrease in Mw reflected the degree of shear degradation of PS molecules during their elution through the SEC columns. It depended on the origins of gels, the size of gel particles, the porosity of frits at column ends, the eluent flow rate, the usage of a pre-column filter, and of the third detector, a capillary viscometer.

A Novel Polar Separation Medium for the Size Exclusion Chromatography of Small Molecules: Uniformly Sized, Porous Poly(vinylphenol-co-divinylbenzene) Beads

A novel separation medium based on uniform size beads of 4-hydroxystyrene-divinylbenzene can be used for both the size exclusion chromatography of small molecules and their separation by reversed-phase chromatography. The new 5 μm material is prepared by the controlled swelling of monodispersed polystyrene particles, that serve both as porogen and shape template, with a polymerization mixture consisting of the monomers and dibutylphthalate, followed by a suspension polymerization. Removal of the acetoxy groups by hydrolysis with aqueous base leads to the final poly(4-vinylphenol-co-divinylbenzene) monodispersed beads. Polymerization conditions that favor the formation of very small pores were developed to optimize the beads for the SEC separation of small molecules. The SEC calibration curve confirms that the optimized beads contain a large volume of pores suitable for the separation of solutes with a molecular weight of up to about 1000. The ability to separate alkylbenzenes according to their hydrodynamic sizes has been demonstrated using a column packed with this material. In addition, the phenol chemistry used in combination with a hydrophobic crosslinking monomer provides this separation medium with an unusual versatility that allows both reversed phase and normal phase chromatography to be run in the same column after a simple change of the mobile phase.

Characterization of an Aqueous-Dissolved Anionic Polysaccharide Using Rheology and Size-Exclusion Chromatography with a Differential Refractive-Index and Low-Angle Laser-Light Scattering Detectors

Abstract From the aqueous growth medium of Red Microalgae Porphyridium sp., anionic polysaccharide was separated by centrifugation and dialysis. Xylose, glucose, galactose, mannose, rhamnose, arabinose, methyl-pentoses, dimethyl-hexoses, glucuronic acid and sulfated sugar-units were identified as constituting monomers. By means of size-exclusion chromatography with differential refractive index and low-angle laser-light scattering (DRI/LALLS) detectors, an average molecular weight of 8.4 × 106 g/mol was determined. For the investigated sample concentrations in the range of 0.08–0.1 mg/mL, SEC separation based on decreasing molecular weight was not observed; however, decreasing particle/fragment dimensions with increasing retention volume were eluted. Rheological investigations at 20°C showed that an aqueous 0.05 M NaCl solution of the dissolved polysaccharide was non-Newtonian with high elasticity. By comparing the polymer-coil packing density of the anionic polysaccharide with three reference polysaccharides, a particle/fragment-packing similar to dextran coils was found. With information about packing density and absolute molecular weight, universal calibration enabled an estimation of dimensions for the constituting components of the anionic polysaccharide in terms of sphere-equivalent radii. The SEC-eluted polymer-coils were found in the range between ≈2–140 nm with a maximum mass occurring at ≈90 nm.

Near-infrared (J, H, K) imaging of Herbig Ae/Be stars

Near-infrared (J, H, K) images were obtained for 16 Herbig Ae/Be stars. The primary goal was to determine the contributions by circumstellar nebulae and nearby sources to near-infrared photometry carried out with large beams. Quasi-simultaneous photometric results were obtained with small apertures. The emission toward five Herbig Ae/Be stars is extended, including all four Group II sources in our sample (Hillebrand et al. 1992); 13 objects have nearby sources (within 10 sec separation). However, the extended emission and nearby sources are too faint to affect previous photometry significantly. The surface brightness profiles of most of the nebulae can be explained by reflection nebulae which scatter the light from the central star/disk systems with single, isotropic scattering processes. The exception is Par 21, which may require emission from very small grains. The color-color diagram, making use of our new photometry, essentially agrees with the results of Lada & Adams (1992). The Group II objects in our sample tend to have extended emission more frequently than do Group I objects, supporting the suggestion of Hillebrand et al. that Group II sources are more affected by circumstellar envelopes. However, most of the near-infrared emission comes from the central (less than or = 6 sec) regions. This upper limit is still much larger than the expected size of accretion disks. Possible envelope effects could not be ruled out for most Herbig Ae/Be stars with unresolved emission. The images do not clearly favor very small, thermally emitting grains as the origin of the near-infrared emission. The problem still exists of how to explain the observed peaks near 3 sec in the spectral energy distributions of Herbig Ae/Be stars. The possible effects of envelopes and companions are addressed.