Patrice Castignolles

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).

Pulsed‐laser radical polymerization and propagation kinetic parameters of some alkyl acrylates

Pulsed-laser photoinitiated polymerization was used to determine, in toluene solution, the propagation kinetic parameters of a series of acrylates with increasing size of the alkyl side group. Transfer to monomer and to toluene did not occur significantly in our PLP conditions and our temperature range since no broadening of the MMD was observed, allowing generally to work with two inflection points. In contrast, depending on the nature of the acrylate and on the PLP conditions, transfer to polymer, and thus long chain branching, can critically interfere. Indeed, the Mark-Houwink-Sakurada parameters, which are used to calculate the absolute molar mass at the inflection point, strongly depend on the polymer structure and thus, should be carefully measured for each PLP sample. Although still preliminary, the results show that the k p s measured in toluene solution present a tendency to continuously decrease when increasing the size of the side group. This observation is conflicting with the reported behaviour for PLP experiments in bulk, revealing a possible solvent effect.

Critically evaluated rate coefficients in radical polymerization – 7. Secondary-radical propagation rate coefficients for methyl acrylate in the bulk

Propagation rate coefficient (kp) data for radical polymerization of methyl acrylate (MA) in the bulk are critically evaluated and a benchmark dataset is put forward by a task-group of the IUPAC Subcommittee on Modeling of Polymerization Kinetics and Processes. This dataset comprises previously published results from three laboratories as well as new data from a fourth laboratory. Not only do all these values of kp fulfill the recommended consistency checks for reliability, they are also all in excellent agreement with each other. Data have been obtained employing the technique of pulsed-laser polymerization (PLP) coupled with molar-mass determination by size-exclusion chromatography (SEC), where PLP has been carried out at pulse-repetition rates of up to 500 Hz, enabling reliable kp to be obtained through to 60 °C. The best-fit – and therefore recommended – Arrhenius parameters are activation energy EA = 17.3 kJ mol−1 and pre-exponential (frequency) factor A = 1.41 × 107 L mol−1 s−1. These hold for secondary-radical propagation of MA, and may be used to calculate effective propagation rate coefficients for MA in situations where there is a significant population of mid-chain radicals resulting from backbiting, as will be the case at technically relevant temperatures. The benchmark dataset reveals that kp values for MA obtained using PLP in conjunction with MALDI-ToF mass spectrometry are accurate. They also confirm, through comparison with previously obtained benchmark kp values for n-butyl acrylate, methyl methacrylate and n-butyl methacrylate, that there seems to be identical family-type behavior in n-alkyl acrylates as in n-alkyl methacrylates. Specifically, kp for the n-butyl member of each family is about 20% higher than for the corresponding methyl member, an effect that appears to be entropic in origin. Furthermore, each family is characterized by an approximately constant EA, where the value is 5 kJ mol−1 lower for acrylates.

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.

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.

Transfer to Polymer and Long-Chain Branching in PLP–SEC of Acrylates

Pulsed laser polymerization (PLP) combined with size exclusion chromatography (SEC) is the method of choice for determining propagation rate coefficients. The influence of the long-chain branching in PLP-SEC is investigated using multiple-detection SEC and a recently developed method to detect long-chain branching [P. Castignolles, R. Grab, M. Parkinson, M. Wilhelm, M. Gaborieau, Polymer 2009, 50, 2373.] While little or no long-chain branching is detected in poly(n-butyl acrylate), the error in relevant molecular weights of poly(2-ethylhexyl acrylate) is large (30-100%) due to long-chain branching. Possible variations of propagation rate coefficient with alkyl groups in alkyl acrylates or with the solvent have to be reconsidered.