Gel Permeation Chromatography Method Research Articles

Analysis of proanthocyanidins by high-performance gel permeation chromatography

A high-performance gel permeation chromatography method was developed for the analysis of proanthocyanidins. The isocratic method consisted of two porous polystyrene–divinylbenzene columns (300×7.5 mm each, 5 μm, 100 and 500 Å individual pore size) and a mobile phase consisting of N, N-dimethylformamide containing 1% (v/v) acetic acid, 5% (v/v) water and 0.15 M lithium chloride. The flow-rate was maintained at 1 ml/min, with a column temperature of 60 °C and with detection at 280 nm. The method was used to analyze proanthocyanidin fractions of increasing molecular mass and from different plant tissues. The average molecular mass of proanthocyanidin fractions as determined by acid catalysis in the presence of phloroglucinol, related well with their gel permeation chromatography column retention, yet significant differences in the retention properties between individual plant tissue isolates existed. Proanthocyanidin compositional differences between isolates may explain these differences. A second-order calibration curve was generated from fractionated grape seed proanthocyanidins and this curve was used to analyze grape seed proanthocyanidins isolated from grapes harvested at extremes of maturity.

Photocurable and thermally stable polymers based on 1,4‐pentadien‐3‐one‐1‐p‐hydroxyphenyl‐5‐p‐phenyl methacrylate: Copolymerization with ethyl acrylate

Abstract1,4‐Pentadien‐3‐one‐1,5‐bis(p‐hydroxyphenyl) (PBHP) was prepared by reacting p‐hydroxybenzaldehyde and acetone in the presence of an acid catalyst. 1,4‐Pentadiene‐3‐one‐1‐p‐hydroxyphenyl‐5‐p‐phenyl methacrylate (PHPPMA) monomer was prepared by reacting PBHP dissolved in ethyl methyl ketone (EMK) with methacryloyl chloride in the presence of triethylamine. A free‐radical solution polymerization technique was used for synthesizing homo‐ and copolymers of different feed compositions of PHPPMA and ethyl acrylate (EA) in EMK as a solvent with benzoyl peroxide as a free‐radical initiator at 70 ± 1 °C. All the polymers were characterized with IR and 1H NMR techniques. The compositions of the copolymers were determined with the 1H NMR technique. The copolymer reactivity ratios were evolved with Kelen–Tudos (EA = 1.25 and PHPPMA = 0.09) and extended Kelen–Tudos (EA = 1.30 and PHPPMA = 0.09) methods. Q (0.48) and e (1.68) values for the new monomer (PHPPMA) were calculated with the Alfrey–Price method. UV absorption spectra for poly(PHPPMA) showed two absorption bands at 302 and 315 nm. The photocrosslinking properties of the polymer samples were examined with the solvent method. Thermal analyses of the polymers were performed with the thermogravimetric‐differential thermogravimetric technique. First, the decomposition temperatures started for poly(PHPPMA), copoly(EA‐PHPPMA) (62:38), and copoly(EA‐PHPPMA) (41:59) were at 350, 410, and 417 °C, respectively. A gel permeation chromatographic method was used for determining the polymer molecular weights (weight‐average molecular weight: 2.67 × 104 and number‐average molecular weight: 1.41 × 104) and polydispersity index (1.89). The solubility of the monomer and the copolymers occurred at 30 °C with solvents having different polarities. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 1632–1640, 2003

Stereospecific Nature of Active Sites on TiCl4/MgCl2 Ziegler–Natta Catalyst in the Presence of an Internal Electron Donor

AbstractBased on our previous study on the donor‐free TiCl4/MgCl2 catalyst, the stereospecific nature of active sites on this catalyst after the addition of an internal donor, ethylbenzoate (EB), was investigated by stopped‐flow propylene polymerization combined with temperature rising elution fractionation (TREF) and gel permeation chromatography (GPC) methods. The addition of donor EB was observed to significantly restrain the formation of aspecific sites (AS sites), to drastically promote formation of sites with the second highest isospecificity (IS2 sites) and to construct some new active sites with the highest isospecificity (IS3 sites). It was first observed that the formation of IS3 sites still strongly depends on the interaction between the catalyst and cocatalyst (up to 10 s of pretreatment) even in the presence of internal donor. Based on Busico and co‐workers' three sites model and our previous report, a plausible mechanism for the transformation of stereospecific active sites through some secondary bimetallic complexation reactions was proposed. The extraction of EB from the catalyst by the cocatalyst was found to initiate after 10 s of pretreatment resulting in partial transformation of IS3 sites into AS sites and IS1 sites. Plausible mechanism for the transformation of active sites with lower isospecificity (AS sites) into the highest isospecific active site (IS3 sites) on the TiCl4/MgCl2 catalyst in the presence of internal donor EB during the pretreatment, X: Cl or Et; M: Mg or Ti. M and Mg are bound to the catalyst substrate through chlorine bridges; □: coordination vacancy.imagePlausible mechanism for the transformation of active sites with lower isospecificity (AS sites) into the highest isospecific active site (IS3 sites) on the TiCl4/MgCl2 catalyst in the presence of internal donor EB during the pretreatment, X: Cl or Et; M: Mg or Ti. M and Mg are bound to the catalyst substrate through chlorine bridges; □: coordination vacancy.

Specific Roles of Al‐Alkyl Cocatalyst in the Origin of Isospecificity of Active Sites on Donor‐Free TiCl4/MgCl2 Ziegler‐Natta Catalyst

AbstractThe formation, deactivation and transformation of stereospecific active sites on the donor‐free TiCl4/MgCl2 catalyst induced by short time reaction with triethylaluminium (TEA) cocatalyst (pretreatment time: 0 ∼ 60 s) were investigated by stopped‐flow propylene polymerization combined with temperature rising elution fractionation (TREF) and gel permeation chromatography (GPC) methods. We got the first report that the origin of isospecificity of active sites derived from the catalyst substrate and cocatalyst were individually observed without and with TEA pretreatment, respectively. Based on the three‐sites model of Corradini and Busico et al., a plausible mechanism of the formation and transformation of stereospecific active sites was proposed using active sites models in terms of specified roles of Al‐alkyl cocatalyst. It was demonstrated that the introduction of bulky alkyl groups instead of chlorine atoms into the neighborhood of active sites seems to be crucial for the generation of active sites with the highest isospecificity. magnified image

Synthesis of multihydroxyl branched polyethers by cationic copolymerization of 3,3‐bis(hydroxymethyl)oxetane and 3‐ethyl‐3‐(hydroxymethyl)oxetane

AbstractThe cationic ring‐opening polymerization of 3,3‐bis(hydroxymethyl)oxetane (BHMO) and the copolymerization of BHMO with 3‐ethyl‐3‐(hydroxymethyl)oxetane (EOX) were studied. Medium molecular weight polymers (number‐average molecular weight ≈ 2 × 103) were obtained in bulk polymerization. Poly[3,3‐bis(hydroxymethyl)oxetane], as highly insoluble, was only characterized by gel permeation chromatography and NMR methods in the esterified form. Copolymers of BHMO and EOX that were slightly soluble in organic solvents were characterized in more detail. In a copolymerization from a 1:1 mixture, the comonomers were consumed at equal rates. Matrix‐assisted laser desorption/ionization time‐of‐flight analysis confirmed that a random 1:1 copolymer was formed. 13C NMR analysis indicated that in contrast to previously described homopolymers of EOX in which the degree of branching was limited, the homopolymers of BHMO were highly branched. This pattern was preserved in the copolymers; EOX units were predominantly linear, whereas BHMO units were predominantly branched. The copolymerization of BHMO with EOX provides, therefore, a route to multihydroxyl branched‐polyethers with various degrees of branching containing OH groups exclusively as ≡CCH2OH units. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 1991–2002, 2002

Two-Dimensional Correlation Gel Permeation Chromatography (2D GPC) Study of 1H,1H,2H,2H-Perfluorooctyltriethoxysilane Sol−Gel Polymerization Process

The novel analytical technique, two-dimensional (2D) correlation analysis, has been applied to a set of time-resolved gel permeation chromatography (GPC) profiles of 1H,1H,2H,2H-perfluorooctyltriethoxysilane (PFOTES) to investigate intricate details of the process of its polymerization, catalyzed by 1 M HCl·H2O, over a thirty minute period. In particular, the splitting behavior of the correlation cross-peaks and their intensity variation have been examined in detail, leading to the conclusion that the mechanism of the growth process of polymeric aggregates is strongly reflected in the patterns of the observed spectra. The 2D correlation GPC method, which has been used to follow the time-dependence of the split components, has proved to be a powerful tool in elucidation of the reaction mechanism.

Flocculation of Humic Substances and Their Derivatives with Chitosan

The efficiency of the flocculation of humic compounds and their derivatives with chitosan was studied as a function of pH, concentrations of chitosan and dissolved humic compounds, and also the presence of inorganic coagulants. Samples of humic compounds were studied by the methods of potentiometric titration and gel permeation chromatography. The molecular weight distributions and the spectra of ionization constants of their functional groups were calculated. The flocculation efficiency of chitosan with respect to humic compounds is much higher than that of polyacrylamide.

Molecular Weight Measurements of Star-Shaped Polystyrene-block-Polybutadiene Copolymers Using Gel Permeation Chromatography

Gel permeation chromatography (GPC) has been used to measure the molecular weight and characterize the number of arms per molecule for star-shaped styrene-butadiene block copolymers (SBS). Conventional GPC method based on polystyrene calibration standards measured a “linear-polystyrene-equivalent” molecular weight that was significantly different from the true molecular weight measured by a GPC hyphenated with a multiangle laser light scattering instrument. The comparison was made on four nearly monodispersed star-shaped copolymers, having a three-arm, four-arm, five-arm, or six-arm molecular structure. In all cases, the “linear-polystyrene-equivalent” molecular weights were higher than the true molecular weights for both the arm and the star molecule. The number of arms as determined based on the “linear-polystyrene-equivalent” molecular weights was consistently smaller than the true number of arms determined based on true molecular weights. The inaccuracy of the conventional GPC method was caused by the use of linear polystyrene calibration standards for characterizing a nonlinear copolymer molecule. The interpolation method developed previously for linear copolymers was unable to produce the accurate molecular weight for star SBS copolymers, and the deviation increases with an increase in the number of arms per molecule.

Photocatalyzed degradation of polymers in aqueous semiconductor suspensions: V. Photomineralization of lactam ring-pendant polyvinylpyrrolidone at titania/water interfaces

The mechanism of photo-oxidation of the water-soluble polyvinylpyrrolidone (PVP) having a pendant five-membered lactam ring is complicated by the manner by which the macromolecule adsorbs on the TiO 2 particle surface in a heterogeneous dispersion. Experimental results and computer simulation of the initial process(es) infer three major steps for the photodegradation of the PVP structure. The first step is adsorption (or coagulation) of PVP on TiO 2 particles as evidenced by the size distribution of TiO 2 particles by dynamic light-scattering and by the electric charge on the TiO 2 particle surface assayed by ζ-potential measurements. Molecular orbital simulations of initial processes were calculated at the AM1 level using the MOPAC system available in the CAChe package. The second step, namely attack of PVP by OH and/or OOH radicals, involves cleavage of the PVP main chain and opening of the lactam ring in the PVP structure probed by temporal UV spectral changes and by a decrease of the molecular weight using gel permeation chromatographic methods. Additional details of the photo-oxidation mode of the lactam ring was examined by a detailed examination of the photo-oxidation of the model compound 2-pyrrolidone to ascertain formation of OH radical adducts, opening of the lactam ring, and identification of intermediates by HPLC, and 13C- and 1H-NMR methods. The final major step in the mechanism involves generation and subsequent conversion of the primary amine (methylamine from opening of the lactam ring) to yield ultimately NH 4 + and NO 3 − ions, and conversion of the propanoic acid to acetic and formic acids and then to CO 2. The effects of the extent of polymerization and variation in light intensity were examined using PVP samples having different hardness factors (hf) of 15 and 30, and different light intensities (namely, 1, 2, 3 and 4 mW cm −2), respectively.

Characterization of high molecular weight cadmium species in contaminated vegetable food.

Spinach and radish grown from seeds were each contaminated with 4 different amounts of cadmium. After a cell breakdown of the eatable parts and centrifugation of the resulting homogenates all supernatants (cytosols) were separated by gel permeation chromatography (GPC). The size-range of the GPC method used was about 20-8000 kDa for globular proteins. The high molecular weight (HMW-Cd-SP, 150-700 kDa) and the low molecular weight Cd species (LMW-Cd-SP, < 150 kDa) in all plant cytosols eluted at about the same retention volume by GPC. The most important Cd binding form in the cytosols of all plants was found to be HMW-Cd-SP. The Cd elution maxima were detected in the range of about 200 kDa. The Cd determinations were performed with ET-AAS by means of matrix modifier. By incubating chosen cytosols with a proteinase before the GPC it was verified that the HMW-Cd-SP in both vegetables are Cd proteins. The molar proportions protein/Cd were about 2-6 in the respective GPC fractions of the HMW-Cd-SP of the highest contaminated plants. The GPC fractions of the HMW-Cd-SP of spinach and radish were further separated by a preparative, native and continuous polyacrylamide gel electrophoresis (PAGE) method. At pH 8 the species were negatively charged, had only a small UV-absorption at 280 nm and showed a very similar elution behavior in all analyzed cytosols. Therefore, we suppose that the HMW-Cd-SP of these two different vegetable foodstuffs have a very similar chemical structure.

Quantitative synthesis and NMR characterization of perhydro-1,3,5-tris(alkylpolyalkoxyl)-S-triazines

Unhindered polyalkoxylated primary amines, upon treatment with an equimolar equivalent of formalde hyde, cyclized quantitatively to afford perhydro-1,3,5-s-triazines. This sereis of derivatives, based upon primary amine terminated poly(propylene and butylene oxides), possessed a molecular weight range of 1,900–5,800. Structure confirmation was accomplished by gel permeation chromatography and 2D nmr spectroscopy methods.

Novel method for separating fatty esters from partial glycerides in biocatalytic transesterification of oils

Abstract Vegetable oil based partial glycerides have been synthesized by lipase catalyzed transesterification of soybean and linseed oil using different primary alcohols. A gel permeation chromatographic method has been used for monitoring the formation of partial glycerides in the transesterification step. A novel laboratory method has been developed for the separation of fatty esters from other partial glycerides by extending the transesterification reaction further by acryloylation/methacryloylation followed by free-radical polymerization. The fatty esters were thus selectively separated by removing the partial glycerides as polymers. Effect of type of lipase, solvent, temperature and substrate to nucleophile ratio in the transesterification step (step I) was also studied. The separation of butyl ester has been confirmed from the structural elucidation by 1H-NMR spectroscopy.

Preparation and Characterization of Highly Water-Soluble Pendant Fullerene Polymers

Pendant fullerene polymers were prepared by attaching methano[60]fullerene carboxylic acid (MFCA) and methano[60]fullerene phenylpropyl carboxylic acid (MFPCA) to linear poly(propionylethylenimine-co-ethylenimine) (PPEI-EI) via amide linkages in carbodiimide-catalyzed coupling reactions. The pendant MFCA-PPEI and MFPCA-PPEI polymers, which were characterized using NMR, gel permeation chromatography, FT-IR, and other instrumental methods, most likely have a charm bracelet type structure without significant cross-linking. MFCA and MFPCA were also coupled with diethylamine in the same carbodiimide-catalyzed reactions, and the methano[60]fullerene amides thus obtained were used as monomeric model compounds for the pendant polymers. UV-vis absorption spectra of the polymer-bound fullerenes, though broader, retain the characteristic features observed in monomeric methano[60]fullerene derivatives. The pendant MFCA-PPEI and MFPCA-PPEI are highly water-soluble, with the equivalent aqueous solubilities of the polymer-bound fullerene cages significantly higher than that of the parent [60]fullerene in room-temperature toluene. Thus, the preparation of pendant fullerene polymers represents an effective approach for introducing fullerene cages into an aqueous solution or hydrophilic environment.

Investigation of the occurrence of pectic-xylan–xyloglucan complexes in the cell walls of cauliflower stem tissues

Selected fractions of cauliflower stem, obtained after ethanol graded precipitation of 0.5 M KOH cell wall extractions, were further purified by anion-exchange and gel-permeation chromatographic methods. Pure xyloglucans and various xylan-rich polysaccharide complexes were obtained, particularly from the most mature section of the stem. A pectic-xylan–xyloglucan complex, tentatively characterised by methylation analysis, was digested separately with purified and highly specific endo-xylanase and also endo-polygalacturonase. Digestions degraded the xylan and pectic polysaccharides moieties, and caused a decrease in the molecular weight of the xyloglucan moieties, thus demonstrating the strong, probably covalent interactions between the different polymer species. The formation of polysaccharide complexes during secondary thickening and the structure of the pectic-xylan–xyloglucan complexes in cauliflower stem tissues are discussed.

Isolation of Soluble Boron Complexes and their Determination Together with Free Boric Acid in Higher Plants

Summary Isolation of soluble boron complexes and their quantitative determination together with free boric acid in higher plants was carried out by gel permeation chromatography in the liquid compartment of plant leaves and roots. The described method,is suitable not only for the isolation of B complexes from plant cell sap, but also allows for their quantitative determination together with free boric acid, which is possible for samples with a B concentration of at least 157#x03BC;molL −1 (Table 2). This gel permeation chromatography method therefore complements methods that allow characterization, but not quantitative determination of B complexes in plant samples. Soluble B complexes have been isolated from sunflower ( Helianthus annuus L.) grown with a marginal to toxic B supply and curly kale ( Brassica oleracea L.). In sunflower root symplasm there was only a small concentration of B complexes that was not dependent on variations in B supply from the marginal to the toxic level. The resulting large variations in total symplasmic B were therefore only reflected in the concentrations of free boric acid. In contrast, the concentration of B complexes in sunflower leaf symplasm was about 50-fold larger than in the root symplasm and made up 31 % of the total symplasmic B. In curly kale leaves even 59 % of the total symplasmic B was present as B complexes. Further experiments are necessary to characterize the B complexes in sunflower and curly kale to investigate their physiological role in plant metabolism.

Electrospray ionization mass spectrometry for the study of non-covalent complexes: an emerging technology.

The detection of non-covalent complexes in the mass range 19,000-34,000 Da, using electrospray ionization mass spectrometry (ESI-MS), is reviewed. The examples discussed include (1) a protein-ligand interaction (ras-GDP), (2) an inhibitor-protein-ligand interaction (SCH 54292/SCH 54341-ras-GDP), (3) a protein-protein interaction (gamma-IFN homodimer) and (4) a protein-metal complex [HCV (1-181)-Zn]. In each case, the ESI-MS method is capable of releasing the intact non-covalent complex from its native solution state into the gas phase in the form of multiply-charge ions. The molecular masses of these complexes were determined with a mass accuracy of better than 0.01%, which is far superior to the traditional methods of sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel permeation chromatography. The method provides the researcher with a quick, reliable and reproducible method for probing difficult biological problems. The key to success in the study of non-covalent complexes depends on careful understanding and manipulation of ESI source parameters and sample solution conditions; special care must be taken with the source orifice potential and the solution pH and organic co-solvents must be avoided. This paper also illustrates the usefulness of ESI-MS for addressing biological problems leading to the discovery of new therapeutics; the approach involves the rapid screening of potential drug candidates, such as weakly bound inhibitors.

Eluent Gel Permeation Chromatography Method for Study of Non-associated Multi-components Solution

Eluent Gel Permeation Chromatography Method for Study of Non-associated Multi-components Solution

Chain folding in cyclic oligo(oxyethylene)s Crystallinity of large unsubstituted crown ethers by X-ray scattering and differential scanning calorimetry

Uniform cyclic oligo(oxyethylene)s with chain lengths E15 to E18 (unsubstituted 45-crown-15 to 54-crown-18) have been prepared by reaction of corresponding linear glycols with tosyl chloride under alkaline conditions, purified by preparative gel permeation chromatography (GPC), and characterised by analytical GPC, NMR spectroscopy, and other methods. Wide-angle and small-angle X-ray scattering were used to investigate crystal structure and layer spacing. Comparison was made with scattering from linear oligomers crystallised in unfolded conformation. It was found that cyclo-E18 crystallised into the poly(oxyethylene) structure (monoclinic sub-cell), while the lower oligomers crystallised into different structures. In confirmation of this result, the layer spacing of cyclo-E18 was that expected for the twice-folded conformation. Melting temperatures and enthalpies of fusion of cyclo-E18 and cyclo-E27 (from previous work), measured by differential scanning calorimetry, were compared with those of linear oligomers to obtain values of the enthalpy and entropy of fold formation in the crystallisation of uniform oligo(oxyethylene).

Probing the Cure and Postcure Reactions in Polyurethanes by FTIR and GPC

Fourier transform infrared (FTIR) and gel permeation chromatography (GPC) wre respectively utilized in determining the changes in isocyanate (NCO) concentration and molecular weight (MW) and its distribution (MWD) in polyurethanes during their cure and postcure stages. Samples, originally cured at 45°C, were taken through accelerate aging tests at various temperatures. The FTIR and GPC analytical conditions were optimized, for both molecular weight and molecular weight distribution and NCO group determination. In FTIR spectroscopy Beer's law was obeyed over the ranges: 2.33-22.34 and 0.03-1.61 mmol lit-1. RSD, detection limit and molar absortivity were found to be 1.46%, 4´10-6 mol lit-1 and 1095 lit mol-1 cm-1 respectively. It was found the NCO concentration decreases with the progress of polymerization and at the same time MW of the sol increases, going through a maximum and then decreases to a constant level. The time to reach this level and termination of NCO concentration, i.e., the end of polymerization decreases with the increase in temperature. The results obtained from GPC and FTIR methods in following the different steps of polymerization as well as those from the linear plots of the end time of reaction vs. the 1/T, were in good agreement.

Measurement and modeling of solvent removal for spin coating

AbstractSolvent concentration is an important factor for spin‐coated film planarization. Two techniques, one an on‐line optical method and the other an off‐line gel permeation chromatography method, have been used to investigate the coating concentration change during spinning. The experimental observations are compared with the spin‐coating model predictions. The modeled concentration and thickness are in close agreement with experiment. Under certain conditions, the spin‐coating model can be significantly simplified. These conditions are discussed.