Marianne Gaborieau

Size-exclusion chromatography (SEC) of branched polymers and polysaccharides

AbstractBranched polymers are among the most important polymers, ranging from polyolefins to polysaccharides. Branching plays a key role in the chain dynamics. It is thus very important for application properties such as mechanical and adhesive properties and digestibility. It also plays a key role in viscous properties, and thus in the mechanism of the separation of these polymers in size-exclusion chromatography (SEC). Critically reviewing the literature, particularly on SEC of polyolefins, polyacrylates and starch, we discuss common pitfalls but also highlight some unexplored possibilities to characterize branched polymers. The presence of a few long-chain branches has been shown to lead to a poor separation in SEC, as evidenced by multiple-detection SEC or multidimensional liquid chromatography. The local dispersity can be large in that case, and the accuracy of molecular weight determination achieved by current methods is poor, although hydrodynamic volume distributions offer alternatives. In contrast, highly branched polymers do not suffer from this extensive incomplete separation in terms of molecular weight. FigureRepresentation of (a) a linear polymer chain and various branched polymer structures with (b) longchain branches (amylose-like), (c) short-chain branches (amylopectin-like), (d) both short-chain and long-chain branches (polyacrylate- or polyethylene-like).

Assessment of the extent of starch dissolution in dimethyl sulfoxide by 1H NMR spectroscopy

Complete dissolution is needed for the separation, characterization, or homogeneous labeling of whole starch molecules. A method is presented to quantify the extent of starch dissolution in DMSO for the first time; it is validated on a commercial rice starch. It is used directly on starch dispersions containing possible undissolved or co-dissolved species. High-amylose maize starches, known to be digested slowly in vivo, only quantitatively dissolve in the presence of high concentrations of an H-bond disrupter, LiBr, although they form clear dispersions at low LiBr concentrations. Starch quantitatively dissolves from waxy rice flours; non-starch components partially co-dissolve but do not interfere with the dissolution quantification.

Mechanistic Investigation of a Starch-Branching Enzyme Using Hydrodynamic Volume SEC Analysis

Two linear alpha-(1,4)-D-glucans substrates, of degrees of polymerization DP approximately 150 and 6000, were exposed to maize starch-branching enzyme IIa (mSBEIIa) in vitro. The resulting branched alpha-glucans and their constituent chains (obtained by debranching) were analyzed by nuclear magnetic resonance (NMR) and size-exclusion chromatography (SEC). SEC data for the debranched species are presented as chain-length distributions, while those for branched species are presented as hydrodynamic volume distributions (HVDs), which is the most meaningful way to present such data (because SEC separates by size, not molar mass, and a sample of branched polymers with the same size can have a range of molar masses). A rigorous interpretation of the HVDs of the substrate and its branched product show that at least part of the branching is an interchain transfer mechanism in both the short- and long-chain substrate cases. A bimodal HVD of the in vitro branched alpha-glucan derived from the short-chain substrate was observed, and it is postulated that the divergence of the two populations is due to very small chains being unable to undergo branching. In the case of the in vitro branching of the long-chain substrate, the formation of maltohexaose during the reaction and the presence of a monomodal HVD were observed, suggesting a distinct mode of action of mSBEIIa on this substrate. Quantification of the branching level by NMR showed the branched glucans from both substrates had substantial amounts of branching (2.1-4.5%), ascribed to the intrinsic nature of the action of mSBEIIa on the two substrates. It is postulated that differences in the degrees of substrate association affect the pattern of branching catalyzed by the enzyme, and a putative active site structure is proposed based on the appearance of maltohexaose. The molar mass distribution of the constituent chains of the in vitro branched alpha-glucans obtained by isoamylase treatment reveals the transfer of chains of specific size and supports the supposition given in the literature that mSBEIIa is responsible for short-chain branching in amylopectin. It is suggested that hydrodynamic volume SEC analysis should be used as a tool for the mechanistic investigation of SBEs, allowing SEC data of in vitro branched alpha-glucans to be both comparable and quantitative.

Toward a full characterization of native starch: Separation and detection by size-exclusion chromatography

The structure of starch molecules is relevant to nutrition and industrial applications. Size-exclusion chromatography (SEC, also known as GPC) of native starch generally suffers non-satisfactory repeatability and reproducibility of the dissolution and separation. This work combines two polar organic solvents: dimethylsulfoxide for complete dissolution and dimethylacetamide to limit shear degradation. The separation is as repeatable as that of polystyrene standards performing dissolution and separation at 80 degrees C. Successful covalent-labeling on the glucose unit is claimed to be published here for the first time in non-degradative conditions and allows the use of UV detector with significantly higher sensitivity than with a refractometer.

Simple and robust determination of monosaccharides in plant fibers in complex mixtures by capillary electrophoresis and high performance liquid chromatography

Carbohydrates partially liberated by acid hydrolysis of plant fiber can be separated by hydrophilic interaction liquid chromatography (HILIC), ligand-exchange liquid chromatography or other forms of LC with ion-exchange columns. However, the robust hydrogen-exchange columns show co-elution of galactose, xylose and mannose. Free solution capillary electrophoresis (CE) can be used without derivatization at pH 12.6 and was found to provide a higher resolution of galactose and xylose than common LC with no sample pre-treatment required, other than dilution, within 26min. CE was able to provide resolution higher than 0.79 for all separated carbohydrates, and the RSDs of determined concentrations lower than 10% for concentrations above 1.3gL(-1). A quantitative comparison between CE and HPLC revealed that up to 22% more carbohydrates are quantified with CE. Direct UV detection in CE of mono- and disaccharides is unexpectedly possible at 270nm. NMR analysis shows that alkaline degradation is too slow to explain this detection. This CE detection sensitivity is increased by the electric field and our CE and NMR analyses are consistent with a photo-oxidation process.

Interplay between structure and dynamics in chitosan films investigated with solid-state NMR, dynamic mechanical analysis, and X-ray diffraction.

Modern solid-state NMR techniques, combined with X-ray diffraction, revealed the molecular origin of the difference in mechanical properties of self-associated chitosan films. Films cast from acidic aqueous solutions were compared before and after neutralization, and the role of the counterion (acetate vs Cl−) was investigated. There is a competition between local structure and long-range order. Hydrogen bonding gives good mechanical strength to neutralized films, which lack long-range organization. The long-range structure is better defined in films cast from acidic solutions in which strong electrostatic interactions cause rotational distortion around the chitosan chains. Plasticization by acetate counterions enhances long-range molecular organization and film flexibility. In contrast, Cl− counterions act as a defect and impair the long-range organization by immobilizing hydration water. Molecular motion and proton exchange are restricted, resulting in brittle films despite the high moisture content.

Synthesis by nitroxide-mediated aqueous dispersion polymerization, characterization, and physical core-crosslinking of pH- and thermoresponsive dynamic diblock copolymer micelles

Diblock copolymers consisting of a poly(acrylic acid) (PAA) segment and a LCST-type poly(N,N-diethylacrylamide) (PDEAAm) block were obtained by nitroxide-mediated polymerization in aqueous dispersion using a water-soluble macroalkoxyamine. The influence of several parameters on the polymerization (temperature, initial free nitroxide or macroalkoxyamine concentrations, and solids content) was evaluated in terms of kinetics, macromolecular control, and colloidal features. As determined by dynamic light scattering (DLS), stable dispersions of monodisperse particles could be obtained for solids content as high as 39 wt% without the need for any additional surfactant via a polymerization-induced self-assembly mechanism. Rendered possible by the use of a controlled/living polymerization process, the effective semi-batch incorporation of hydrophobic units (styrene) in the growing chains during the polymerization allowed the formation of physically crosslinked nanogels. The pH and temperature sensitivity were proved by means of DLS and high-sensitivity differential scanning calorimetry (HSDSC) measurements. Due to the formation of aggregates observed by size-exclusion chromatography in N,N-dimethylformamide, accurate molar masses could not be determined directly but deconvoluted hydrodynamic volume distributions suggested a good control of the polymerization.

Heterogeneous modification of chitosan via nitroxide-mediated polymerization

Chitosan (CS) was modified by SG1-based nitroxide-mediated polymerization under heterogeneous conditions. After introduction of acrylamide and/or acrylate functions onto the CS backbone followed by intermolecular 1,2 radical addition of the BlocBuilder alkoxyamine (CS–BB), methyl methacrylate (MMA) in the presence of a small amount of acrylonitrile (AN) or sodium 4-styrenesulfonate (SS) was polymerized by nitroxide-mediated polymerization (NMP). ESR and free-solution capillary electrophoresis confirmed the synthesis of CS–BB. The successful synthesis of CS-g-P(MMA-co-AN) and CS-g-PSS grafted copolymers was proved by TGA and solid-state NMR spectroscopy with ca. 20 to 30 wt% of grafted synthetic polymer in the final product.

Separation of chitosan by degree of acetylation using simple free solution capillary electrophoresis

AbstractChitosan is a biopolymer of increasing significance, as well as a renewable and sustainable material. Its main molecular characteristics are molar mass and degree of acetylation (composition). Precise average degrees of acetylation were measured by quantitative 1H solution-state NMR spectroscopy. While number-average degrees of acetylation had already been determined by 1H NMR spectroscopy, weight-average degrees of acetylation are also determined and may be more relevant for some properties, such as mechanical properties. We report the first separation of chitosan according to its degree of acetylation using free solution capillary electrophoresis. Capillary electrophoresis separates chitosan in the ‘critical conditions’: the molar mass plays little role and the separation is by the degree of acetylation. It characterises the heterogeneity of chitosan samples in terms of composition (dispersity of the distribution of degrees of acetylation). This heterogeneity (broad distribution of degrees of acetylation) cannot be neglected contrary to a common assumption found in the literature. This fast and easy separation will allow establishing a structure–property relationships. FigureSeparation of chitosan molecules by their composition with free solution capillary electrophoresis

Green nondegrading approach to alkyne-functionalized cellulose fibers and biohybrids thereof: synthesis and mapping of the derivatization.

Alkyne-functionalized cellulose fibers have been generated through etherification under basic water or hydroalcoholic conditions (NaOH/H(2)O/isopropanol). For a given NaOH content, the medium of reaction and, more particularly, the water/IPA ratio, were shown to be of crucial importance to derivatize the fibers without altering their integrity and their crystalline nature. It was shown that the degree of substitution (DS) of the fibers increases concomitantly with isopropanol weight ratio and that, contrary to water or water-rich conditions, derivatization of fibers under isopropanol-rich conditions induces an alteration of the fibers. Optimization of etherification conditions in aqueous media afforded functionalized cellulose materials with DS up to 0.20. Raman confocal microscopy on derivatized fibers cross sections stressed that alkyne moieties are incorporated all over the fibers. The resulting fibers were postfunctionalized by molecular probes and macromolecules in aqueous or water-rich conditions. The effectiveness of the grafting was strongly impacted by the nature of the coupling agents.