Coupling Size Exclusion Chromatography Research Articles

On-line size exclusion chromatography–pyrolysis-gas chromatography–mass spectrometry for copolymer characterization and additive analysis

On-line coupled size exclusion chromatography–pyrolysis gas chromatography mass spectrometry (SEC–Py-GC–MS) is studied as a novel tool for the characterization of complex polymer samples. An automated system for on-line SEC–Py-GC–MS allowing transfer of multiple fractions was developed based on stop-flow operation of the SEC dimension, syringe-based transfer of the SEC fraction to the GC instrument and solvent elimination with subsequent pyrolysis in a programmed temperature vaporization (PTV) injector. After optimization the system was applied to the characterization of a complex terpolymer composed of very similar monomers. The use of the system for combined pyrolysis and additive analyses in polycarbonate was also demonstrated. Results obtained with the new method indicate the interesting potentials of the method for detailed characterization of polymeric materials.

Surface-Enhanced Raman Spectroscopy of Chernozem Humic Acid and Their Fractions Obtained by Coupled Size Exclusion Chromatography—Polyacrylamide Gel Electrophoresis (SEC-PAGE)

A humic acid extracted from a chernozem soil was fractionated combining size exclusion chromatography and polyacrylamide electrophoresis (SEC-PAGE). Three fractions named A, B, and C+D, with different electrophoretic mobilities and molecular sizes (MS), were obtained and subsequently characterized by thermochemolysis and surface-enhanced Raman spectroscopy (SERS). The data confirmed that fraction A, with the higher MS, was more aliphatic than fractions B and C+D and, in turn, fractions with lower MS (B and C+D) denoted an enrichment in lignin residues. These structural features explain conformational changes when varying the pH in the humic fraction A and indicated that combination of the two techniques is a good approach for characterizing humic substances.

An in-depth analytical approach to the mechanism of the RAFT process in acrylate free radical polymerizations via coupled size exclusion chromatography–electrospray ionization mass spectrometry (SEC–ESI-MS)

Coupled size exclusion chromatography (SEC)–electrospray ionization mass spectrometry (ESI-MS) was applied to carefully map the product spectrum of a series of acrylate free radical polymerizations mediated via the reversible addition fragmentation chain transfer (RAFT) process. The product stream of a significantly rate retarded RAFT system (i.e. n-butyl acrylate (BA)/cumyl dithiobenzoate (CDB)) was compared with the less rate retarded RAFT polymerizations of BA mediated by cumyl phenyl dithioacetate (CPDA) and methyl acrylate (MA)/CPDA. In each case excellent agreement between the theoretical and experimental masses, as well as the simulated isotopic peak distributions, of polymeric species in the product stream was observed. Although conventional disproportionation and combination bimolecular termination products were clearly identified within the product spectra, the presence of irreversibly terminated RAFT intermediates, i.e. 3-armed star polymers, was not observed. The mass spectroscopic results are compared to modeling estimations (carried out via the PREDICI® program package) of the concentration ratios of 3-armed stars vs. conventional termination products. It is demonstrated that the occurrence of conventional termination products should be accompanied by a significant product stream associated with 3-armed star polymer material if cross termination was operational—at least under the current reaction conditions. The absence of three armed star polymer products in the polymers stream suggests that irreversible cross termination reactions may be of minor importance in the present systems.

SEC-MALS method for the determination of long-chain branching and long-chain branching distribution in polyethylene

This paper systematically describes a LCB determination method that can quantify both LCB content and LCB distribution across the molecular weight distribution in polyethylene homopolymers as well as copolymers. Coupling size-exclusion chromatography with multi-angle light scattering (SEC-MALS), this method quantifies molecular weights (MW) and radii of gyration ( R g) simultaneously. The number of LCB per molecule and LCB frequency as a function of MW can be calculated by comparing R g of a branched polymer with that of a linear control at the same MW using the Zimm–Stockmayer approach. Because the presence of short-chain branching in copolymers results in changes in R g of the copolymers, their LCB contents cannot be obtained before the short-chain branching (SCB) effect is corrected. Using well-characterized linear PE copolymers as standards, an empirical method is successfully established in this paper to correct the SCB effect. Consequently, this method can be applied to determine LCB in PE copolymers as well. Some practical aspects, such as the selection of formalism for data processing, the LCB detection sensitivity and precision, and long-term reproducibility of this method are also discussed. Finally, examples are given to demonstrate how this method is applied to determine LCB and LCB distribution in practical PE homopolymers and copolymers.

Thermochemolysis of genetically different soil humic acids and their fractions obtained by tandem size exclusion chromatography–polyacrylamide gel electrophoresis

Humic acids (HA) from soils of very different origin and bioclimatic conditions (podzoluvisol, phasozem, chernozem, and ferralsol) were fractionated by coupling size-exclusion chromatography and polyacrylamide gel electrophoresis (tandem SEC–PAGE). From each HA, three fractions—A, B, and C+D, with different molecular sizes and electrophoretic mobilities—were obtained and analysed by thermochemolysis, the products being identified subsequently by gas chromatography/mass spectrometry. Regardless of HA origin, fraction A contained considerably more n-fatty acids and n-alkanes derived from plant waxes than did fractions B and C+D. Lignins accumulated mainly in fractions B and C+D. These results show that tandem SEC–PAGE allowed the separation of soil HA into fractions enriched in different structural components whose distribution was independent of HA genesis.

Investigation of the elemental composition and chemical association of several elements in fulvic acids dietary supplements by size-exclusion chromatography UV inductively coupled plasma mass spectrometric

Four fulvic acid dietary supplement samples were obtained for this study with the intention of investigating the elemental composition and association of fulvic acids found in fulvic acid supplements. This was achieved by coupling size-exclusion chromatography (SEC) sequentially with UV–vis and inductively coupled plasma mass spectrometric (ICP-MS) detectors. The combination of UV and ICP-MS offered highly sensitive and selective detection. This technique was used in the present study to initially investigate the chemical association of several different elements including, Cr, Co, Ca, Fe, I, Mg, Zn, Se, Cu, Mn, Mo, As, Hg, Pb, and Ag, by observing the elution profile of the fulvic acids obtained with UV detection and matching their retention times with the peaks measured with ICP-MS. The results found based on this type of analysis suggest that there was some association of the elements to the fulvic acids. It was also of interest to observe the stability of these complexes upon human digestion; therefore a gastric digestion was mimicked. In the fulvic acid dietary supplement samples studied, fulvic acids were present in the samples and there was elemental association based on the retention time overlap in the UV as well as the ICP-MS. The fulvic complexes found in the samples were of a low molecular weight As a result of the digestion the SEC–ICP-MS chromatographic profile in some of the samples changed, which may infer that the elemental association had changed.

Investigation of the elemental composition and chemical association of several elements in fulvic acids dietary supplements by size-exclusion chromatography UV inductively coupled plasma mass spectrometric

Four fulvic acid dietary supplement samples were obtained for this study with the intention of investigating the elemental composition and association of fulvic acids found in fulvic acid supplements. This was achieved by coupling size-exclusion chromatography (SEC) sequentially with UV–vis and inductively coupled plasma mass spectrometric (ICP-MS) detectors. The combination of UV and ICP-MS offered highly sensitive and selective detection. This technique was used in the present study to initially investigate the chemical association of several different elements including, Cr, Co, Ca, Fe, I, Mg, Zn, Se, Cu, Mn, Mo, As, Hg, Pb, and Ag, by observing the elution profile of the fulvic acids obtained with UV detection and matching their retention times with the peaks measured with ICP-MS. The results found based on this type of analysis suggest that there was some association of the elements to the fulvic acids. It was also of interest to observe the stability of these complexes upon human digestion; therefore a gastric digestion was mimicked. In the fulvic acid dietary supplement samples studied, fulvic acids were present in the samples and there was elemental association based on the retention time overlap in the UV as well as the ICP-MS. The fulvic complexes found in the samples were of a low molecular weight As a result of the digestion the SEC–ICP-MS chromatographic profile in some of the samples changed, which may infer that the elemental association had changed.

Reversible Addition Fragmentation Chain Transfer (RAFT) Polymerization of Methyl Acrylate: Detailed Structural Investigation via Coupled Size Exclusion Chromatography−Electrospray Ionization Mass Spectrometry (SEC−ESI-MS)

The on-line coupling of size exclusion chromatography (SEC) with electrospray ionization mass spectrometry (ESI-MS) was applied to study the polymeric product spectrum generated by the (strongly rate retarded) 2,2‘-azobisisobutyronitrile (AIBN) and 1,1‘-azobis(cyclohexanecarbonitrile) (ACHCN) initiated, cumyl dithiobenzoate (CDB) mediated methyl acrylate (MA) polymerization in detail. Products corresponding to the polymeric reversible addition fragmentation chain transfer (RAFT) agent and the combination and disproportionation termination products (with both cumyl, cyanocyclohexyl, and cyanoisopropyl end groups) along with a product stream arising from an oxidation process of the polymeric RAFT agent yielding sulfines and thioesters could be clearly identified. Termination products associated with the intermediate radical, that is, three- or four-armed star polymers, were not present. There is a degree of ambiguity over the disproportionation product of the macroRAFT radical. The experiments suggest that either the RAFT intermediate disproportionation product or the actual RAFT intermediate radical is present in the ESI-MS spectrum.

Quantitative characterization of a polystyrene/poly(alpha-methylstyrene) blend by MALDI mass spectrometry and size-exclusion chromatography.

The use of coupled size-exclusion chromatography and MALDI mass spectrometry for quantitative measurement of the composition of technical polymer blends at the molecular level is described. The method is illustrated with a model binary blend consisting of polystyrene and poly(alpha-methylstyrene), both polymers having similar molecular weights and polydispersities. The proposed MALDI data treatment allows determination of the chemical composition of the blend, the molecular weights of the constituents, and the distributions of the homopolymers.

Capillary zone electrophoresis of soil humic acid fractions obtained by coupling size-exclusion chromatography and polyacrylamide gel electrophoresis

Capillary zone electrophoresis (CZE) was used for characterisation of soil humic acid (HA) fractions obtained by coupling size-exclusion chromatography with polyacrylamide gel electrophoresis, on the basis of their molecular size and electrophoretic mobility. CZE was conducted using several low alkaline buffers as background electrolyte (BGE): 50 m M carbonate, pH 9.0; 50 m M phosphate, pH 8.5; 50 m M borate, pH 8.3; 50 m M Tris–borate+1 m M EDTA+7 M urea+0.1% sodium dodecyl sulphate (SDS), pH 8.3. Independently of BGE conditions, the effective electrophoretic mobility of HA fractions were in good agreement with their molecular size. The better resolution of HA were obtained in Tris–borate–EDTA buffer with urea and SDS. This results indicated that CZE, mostly with BGE-contained disaggregating agents, is useful for separating HAs in fractions with different molecular sizes.

On-line coupling of SEC and RP–LC for the determination of structurally related enkephalins in cerebrospinal fluid

On-line coupled analytical techniques can be advantageous in the assay of smaller peptides in complex biological matrices such as plasma, cerebro-spinal fluid (CSF) and tissues. The present study shows the feasibility of the recently reported on-line coupled size exclusion chromatography (SEC) and reversed phase liquid chromatography (RP–LC) separation system for the quantitation of structural related peptides in biological matrices, as demonstrated for a number of enkephalins in CSF. The degradation of the peptides, caused by endogenous peptidases in the matrix, could sufficiently be inhibited with imipramine HCL to allow an assay with satisfactory linearity and intraday (0.70–4.9%) precision. The sensitivity of the method, with a concentration limit of quantitation (LOQ) of 2 μg/ml is comparable with other kinds of assays for peptides and sufficient for the quantitation of peptide drugs with higher therapeutic ranges in biological matrices. However, for the assay of low concentrations of peptides, such as endogenous components of a biological matrix, the sensitivity may need improvement. The LOQ cannot be improved by increasing the sample amount, because of interference of other endogenous components of the CSF. This indicates that a larger selectivity is desired. The LOQ may be improved by using more sensitive and selective detection methods such as mass spectrometry or fluorescence after post-column derivatization. Miniaturization of the system, combined with on-line trapping may also contribute to a better sensitivity.

Influence of long-chain branches in polyethylenes on linear viscoelastic flow properties in shear

This contribution presents a survey on the influence of long-chain branching on the linear viscoelastic properties zero shear-rate viscosity and steady-state recoverable compliance of polyethylene melts. The materials chosen are linear and slightly long-chain branched metallocene-catalyzed polyethylenes of narrow molecular mass distribution as well as linear and highly long-chain branched polyethylenes of broad molecular mass distribution. The linear viscoelastic flow properties are determined in shear creep and recovery experiments by means of a magnetic bearing torsional creep apparatus. The analysis of the molecular structure of the polyethylenes is performed by a coupled size exclusion chromatography and multi-angle laser light scattering device. Polyethylenes with a slight degree of long-chain branching exhibit a surprisingly high zero shear-rate viscosity in comparison to linear polyethylenes whereas the highly branched polyethylenes have a much lower viscosity compared to linear samples. Slightly branched polyethylenes have got a higher steady-state compliance in comparison to linear products of similar polydispersity, whereas the highly branched polyethylenes of broad molecular mass distribution exhibit a surprisingly low elasticity in comparison to linear polyethylenes of broad molecular mass distribution. In addition sparse levels of long-chain branching cause a different time dependence in comparison to linear polyethylenes. The experimental findings are interpreted by comparison with rheological results from literature on model branched polymers of different molecular topography and chemical composition.

NOM-facilitated transport of metal ions in aquifers: importance of complex-dissociation kinetics and colloid formation

The transport of metal ions (Al, Fe, Zn, Pb) complexed by natural organic matter (NOM) was investigated by column experiments. Direct breakthrough of metal–NOM complexes was observed after elution of one bed volume for Al, Fe, and Pb, but not for Zn. This observation cannot be understood assuming local thermodynamic equilibrium in the columns. Hence, a model was developed, taking into account the kinetics of the interactions of metal ions with NOM and the solid phase. Of all possible reactions during passage through the column, the dissociation of the metal–NOM complexes was assumed to be the rate-determining step. Dissociation-rate constants were determined by cation-exchange experiments with a non-NOM-adsorbing cation-exchange resin (Chelex-100). These rate constants were used to predict the migration of metal–NOM complexes in the column experiments. Experimental and modeling results were in good agreement for the bivalent metal ions. Also, the model well predicted the pH dependence of breakthrough of trivalent metal ions and the differences between Al and Fe breakthrough on a qualitative basis. This leads to the conclusion that the dissociation kinetics of metal–NOM complexes is an essential parameter for the estimation of NOM-facilitated metal transport. However, for the trivalent metals, Al and Fe, the model overestimated the direct breakthrough, thus giving a worst case prediction of metal transport. With the help of coupling size-exclusion chromatography to inductively coupled plasma–mass spectrometry, the formation of Al-hydroxide and Fe-hydroxide colloids in addition to NOM complexes was detected. These colloids, which were not considered in the model, were partially filtered off in the column, thus leading to overestimation of metal breakthrough.

Sec-Page Characterization of Lake Aquatic Humic Matter Isolated with Xad-Resin and Tangential Membrane Ultrafiltration

Several aquatic heterogeneous humic-solute fractions with known molecular-size ranges were fractionated further by coupled size-exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE). The results of SEC-PAGE, obtained for several humic-solute fractions, corresponded reasonably well to the known molecular-size distributions of the dissolved organic matter (DOM). Despite the limited resolving power PAGE appears to offer an alternative coarse method, beside SEC, for fractionation of the heterogeneous humic matter (HM).

Photoinductive properties of soil humic acids and their fractions obtained by tandem size exclusion chromatography–polyacrylamide gel electrophoresis

Humic acids (HAs) from three soils of different origin (Chernozem, Ferralsol and Ranker) have been fractionated by coupling size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE) on three fractions (fractions A, B, C + D) with different molecular sizes (MSs) and exactly defined electrophoretic mobility (EM). Fractions identically marked had similar EM and MS, independently of HA sources. The photoinductive properties of the whole HAs and their fractions were compared by studying the photoinduced transformation of fenuron at 365 nm. High MS fractions A and B appeared to exhibit poor photoinductive activities compared to the whole HAs, whereas low MS fraction C + D in Chernozem and Ranker were more efficient than the whole HAs. A fourth intermediary fraction containing a mixture of fractions B and C + D with small amount of D was shown to photoinduce poorly the transformation of fenuron. It was therefore concluded that the molecules capable of photoinducing the transformation of fenuron were mainly contained in fraction D. Fluorescence properties of Chernozem HA and its fractions have been tested. Fraction C + D exhibited a very similar fluorescence emission spectrum in comparison with the whole HA and in contrast, the fractions A and B emitted very weakly.

MS/NMR: a structure-based approach for discovering protein ligands and for drug design by coupling size exclusion chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy.

A protocol is described for rapidly screening small organic molecules for their ability to bind a target protein while obtaining structure-related information as part of a structure-based drug discovery and design program. The methodology takes advantage of and combines the inherent strengths of size exclusion gel chromatography, mass spectrometry, and NMR to identify bound complexes in a relatively universal high-throughput screening approach. Size exclusion gel chromatography in the spin column format provides the high-speed separation of a protein-ligand complex from free ligands. The spin column eluent is then analyzed under denaturing conditions by electrospray ionization mass spectrometry (MS) for the presence of small molecular weight compounds formerly bound to the protein. Hits identified by MS are then individually assayed by chemical shift perturbations in a 2D 1H-15N HSQC NMR spectrum to verify specific interactions of the compound with the protein and identification of the binding site on the protein. The utility of the MS/NMR assay is demonstrated with the use of the catalytic fragment of human fibroblast collagenase (MMP-1) as a target protein and the screening of a library consisting of approximately 32 000 compounds for the identification of molecules that exhibit specific binding to the RGS4 protein.

Evaluation of the transformation of organic matter to humic substances in compost by coupling sec-page

Humic acids (HAs) from soil and compost at the beginning (S0) and at the end of the stabilization process after 130 days (S130) have been fractionated by coupling size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE). Preparative quantities of HA fractions (HAFs) with different molecular sizes (MSs) and exactly defined electrophoretic mobility (EMs) have been obtained from all samples and the HAFs weight content has been studied. A high degree of similarity in HAFs weight content between soil HA and a stabilized compost HA S130 has been observed. Such data seem to be reliable for monitoring the evolution of the compost organic matter to humic substances for their agricultural uses.

Discrimination of Poly(ethyl methacrylate)s According to Their Molar Mass and Tacticity by Coupling Size Exclusion Chromatography and Liquid Chromatography at the Critical Adsorption Point

A coupling of size exclusion chromatography (SEC) and liquid chromatography at the critical adsorption point (LC CAP) has been applied to simultaneous discrimination of stereoregular poly(ethyl methacrylate)s (PEMAs) according to their molar mass and tacticity. The two-dimensional SEC/LC CAP separations were carried out with a chromatographic system which consisted of two analytical columns, one SEC column (polystyrene/divinylbenzene gel) and one LC CAP column (aminopropyl bonded silica gel) connected in series. Narrow fractions of PEMA samples eluted from the SEC column were on-line forwarded into the LC CAP column to be separated according to their tacticity. The column system was flushed with tetrahydrofuran (THF)/cyclohexane mixed eluents at compositions of 64/36 and 56/44 w/w, to attain the molar-mass-independent elution of either highly syndiotactic or highly isotactic PEMAs, respectively. In these eluents, the SEC column still separated PEMAs solely according to their molar masses, while the LC CAP...

Weight and optical differences between soil humic acids fractions obtained by coupling SEC-PAGE

Humic acids (HAs) from four soils of different origin (podzoluvisol, phasozem, chernozem and ferralsol) have been fractionated by coupling size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE). Preparative quantities of HA fractions (HAFs) with different molecular sizes (MSs) and exactly defined electrophoretic mobility (EM) have been obtained from all the samples. Fractions identically marked had similar EM, independently of HA sources. The weight content, extinction coefficients (ECs), E4/E6 ratios have been studied for all HAFs. EC values and E4/E6 ratios increased as MS decreased within an individual HA sample. The weight content of high MS fraction was the highest in podzoluvisol and ferralsol and the smallest in phasozem and chernozem. On the other hand, chernozem and phasozem HAs contained considerably more of the lower MS fraction than HAs from the podzoluvisol and ferralsol. The knowledge of weight and optical differences between soil humic acids fractionated by coupling SEC-PAGE could be valued for explanation of some physico-chemical properties of soils and influence of soil genesis on humic matter properties.

Separation of metalloprotein complexes in serum by size-exclusion chromatography: Optimisation of the separation parameters retention behaviour and recovery employing radiotracers

A suitable procedure was developed for speciation analysis of metalloprotein complexes in serum using directly coupled size-exclusion chromatography and an element-specific detector. Two column matrices used for size-exclusion chromatography (TSK G 3000 SW and Asahipak GS 520) were investigated with respect to the recovery and retention behaviour for metalloprotein complexes. Optimisation of the separation parameters (buffer type, concentration, pH) was achieved by means of metalloprotein complexes marked with radiotracers. For speciation of serum the matrix in the Asahipak GS column is more efficient. Given optimal eluent characteristics (100 m M Tris, pH 7.4) the recovery of the elements investigated (sodium, calcium, iron and zinc) was 100%. Further, the retention behaviour (retention time, ratios of the peak areas) remained unchanged for several successive separations.