Using Capillary Electrophoresis to Investigate Protein Conformational and Compositional Heterogeneity.

Detailed insights into protein structure/function relationships require robust characterization methodologies. Free-solution capillary electrophoresis (CE) is a unique separation technique which is sensitive to the conformation and/or composition of proteins, and therefore provides information on the heterogeneity of these properties. Three unrelated, conformationally/compositionally-altered proteins were separated by CE. An electrophoretic mobility distribution was determined for each protein along with its conformational and/or compositional heterogeneity. The CE results were compared with molar mass distributions obtained from size-exclusion chromatography coupled to light scattering (SEC-MALS). Bovine serum albumin multimers and two monomeric species were separated, highlighting variations in conformational/compositional heterogeneity among the multimers. Analysis of yeast alcohol dehydrogenase resolved two monomeric conformers and various tetrameric species, illustrating the impact of zinc ion removal and disulfide bond reduction on the protein's heterogeneity. The apo (calcium-free) and holo forms of bovine α-lactalbumin were separated and differences in the species' heterogeneity were measured; by contrast, the SEC-MALS profiles were identical. Comparative analysis of these structurally unrelated proteins provided novel insights into the interplay between molar mass and conformational/compositional heterogeneity. Overall, this study expands the utility of CE by demonstrating its capacity to discern protein species and their heterogeneity, properties which are not readily accessible by other analytical techniques.

Insight into Three-Coordinate Aluminum Species on Ethanol-to-Olefin Conversion over ZSM-5 Zeolites.

Commercial bioethanol can be readily converted into ethylene by a dehydration process using solid acids, such as Brønsted acidic H-ZSM-5 zeolites, and thus, it is an ideal candidate to replace petroleum and coal for the sustainable production of ethylene. Now, strong Lewis acidic extra-framework three-coordinate Al3+ species were introduced into H-ZSM-5 zeolites to improve their catalytic activity. Remarkably, Al3+ species working with Brønsted acid sites can accelerate ethanol dehydration at a much lower reaction temperature and shorten the unsteady-state period within 1-2 h, compared to >9 h for those without Al3+ species, which can significantly enhance the ethanol dehydration efficiency and reduce the cost. The reaction mechanism, studied by solid-state NMR, shows that strong Lewis acidic EFAl-Al3+ species can collaborate with Brønsted acid sites and promote ethanol dehydration either directly or indirectly via an aromatics-based cycle to produce ethylene.

Poly(ethylene glycol) functionalization of monolithic poly(divinyl benzene) for improved miniaturized solid phase extraction of protein-rich samples.

Non-specific protein adsorption on hydrophobic solid phase extraction (SPE) adsorbents can reduce the efficacy of purification. To improve sample clean-up, poly(divinyl benzene) (PDVB) monoliths grafted with hydrophilic polyethylene glycol methacrylate (PEGMA) were developed. Residual vinyl groups (RVGs) of the PDVB were employed as anchor points for PEGMA grafting. Two PEGMA monomers, M n 360 and 950, were compared for graft solutions containing 5-20% monomer. Protein binding was qualitatively screened using fluorescently labeled human serum albumin (HSA) to determine optimal PEGMA concentration. The fluorescent signal of PDVB was reduced for PDVB-g-PEGMA360 (10%) and PDVB-g-PEGMA950 (20%). The PEGMA content (w/w%) was quantified by solid state 1H NMR to be 29.9 ± 1.6% for PDVB-g-PEGMA360 and 7.7 ± 1.2% for PDVB-g-PEGMA950. To assess adsorbent performance breakthrough curves for PDVB, PDVB-g-PEGMA360 and PDVB-g-PEGMA950 were compared. The breakthrough volume (V B) and shape of the curve for PDVB-g-PEGMA950 were maintained relative to PDVB (2.3 and 2.8 mL, respectively). A reduced V B of 0.5 mL and shallow breakthrough curve indicated PDVB-g-PEGMA360 was not suitable for SPE. A high ibuprofen recovery of 92 ± 0.30 and 78 ± 0.93% was seen for PDVB and PDVB-g-PEGMA950, respectively. Protein adsorption was reduced from 31 ± 2.41 to 12 ± 0.49% for PDVB and PDVB-g-PEGMA950, respectively. SPE of ibuprofen from plasma was compared for PDVB and PDVB-g-PEGMA950 by at-line electrospray ionization mass spectrometry (ESI-MS). PDVB-g-PEGMA950 demonstrated a threefold increase in assay sensitivity indicating a superior analyte purification.

Quantifying the Heterogeneity of Chemical Structures in Complex Charged Polymers through the Dispersity of Their Distributions of Electrophoretic Mobilities or of Compositions.

The complexity of synthetic and natural polymers used in industrial and medical applications is expanding; thus, it becomes increasingly important to improve and develop methods for their molecular characterization. Free-solution capillary electrophoresis is a robust technique for the separation and characterization of both natural and synthetic complex charged polymers. In the case of polyelectrolytes, free-solution capillary electrophoresis is in the "critical conditions" (CE-CC): it allows their separation by factors other than molar mass for molar masses typically higher than 20000 g/mol. This method is thus complementary to size-exclusion chromatography (SEC). SEC is widely used to determine molar mass distributions and their dispersities. Utilizing CE-CC, an analogous calculation of dispersity based on the distributions of electrophoretic mobilities was derived and the heterogeneity of composition or branching in different polysaccharides or synthetic polymers was obtained in a number of experimental cases. Calculations are based on a ratio of moments and could therefore be compared to simulations of polymerization processes, in analogy to the work performed on molar mass distributions. Among four possible types of dispersity, the most precise values were obtained with the calculation analogous with the dispersity of molar mass distribution Mw/Mn. In addition, the dispersity value allows conclusions based on a single value: the closer the dispersity is to 1, the more homogeneous the polymer is in terms of composition or branching. This approach allows the analysis of dispersity of important molecular attributes of polymers other than molar mass and aims at improving the overall molecular characterization of both synthetic and natural polymers. The dispersity can also be monitored online while performing a chemical reaction within the CE instrument.

Structural modifications of cellulose samples after dissolution into various solvent systems.

This work deals with the modifications resulting from the dissolution of four commercial cellulosic samples, with different crystallinity rates and degrees of polymerization (DPs), in four solvent systems, known and used to dissolve cellulose. The dissolution conditions were optimized for the 16 various systems and followed by turbidity measurements. After regeneration, the samples were analyzed by thermal gravimetric analysis (TGA), scanning electron microscopy (SEM), and X-ray diffractometry (XRD) to study their modification. Viscosimetry measurements were used to evaluate the potential decrease of the DP after dissolution. The observed structural modifications established that, for low DP, all the solvent systems were efficient in dissolving the cellulose without altering the DP, except BMIM [Cl], which provoked a decrease of up to 40 % and a decrease of around 20 % of the degradation temperature (onset temperature, T o). For high molecular weight (MW) celluloses, DMSO/TBAF was the only system to allow a complete dissolution without any molar mass loss and degradation temperature modification.

Real-time monitoring of peptide grafting onto chitosan films using capillary electrophoresis.

Chitosan, being antimicrobial and biocompatible, is attractive as a cell growth substrate. To improve cell attachment, arginine-glycine-aspartic acid-serine (RGDS) peptides were covalently grafted to chitosan films, through the widely used coupling agents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC-HCl) and N-hydroxysuccinimide (NHS), via the carboxylic acid function of the RGDS molecule. The grafting reaction was monitored, for the first time, in real time using free-solution capillary electrophoresis (CE). This enabled fast separation and determination of the peptide and all other reactants in one separation with no sample preparation. Covalent RGDS peptide grafting onto the chitosan film surface was demonstrated using solid-state NMR of swollen films. CE indicated that oligomers of RGDS, not simply RGDS, were grafted on the film, with a likely hyperbranched structure. To assess the functional properties of the grafted films, cell growth was compared on control and peptide-grafted chitosan films. Light microscopy and polymerase chain reaction (PCR) analysis demonstrated greatly improved cell attachment to RGDS-grafted chitosan films.

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.

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

Chitosan 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 (1)H solution-state NMR spectroscopy. While number-average degrees of acetylation had already been determined by (1)H 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.

Separation of poly(acrylic acid) salts according to topology using capillary electrophoresis in the critical conditions.

Branching was detected in polyacrylates synthesised through radical polymerization via solution-state NMR, while inconsistencies have been reported for the determination of the molar mass of hydrophilic polyacrylates using aqueous-phase and organic-phase size-exclusion chromatography. In this work, poly(sodium acrylate)s, PNaAs, of various topologies were separated for the first time using free-solution capillary electrophoresis (CE). Free-solution CE does not separate the PNaAs by their molar mass, similarly to separations by liquid chromatography in the critical conditions, rather by different topologies (linear, star branched, and hyperbranched). The electrophoretic mobility of PNaAs increases as the degree of branching decreases. Separation is shown to be not only by the topology but also by the end groups as expected for a separation in the critical conditions: replacing a relatively bulky nitroxide end group with hydrogen atom yielded a higher electrophoretic mobility. This novel method, capillary electrophoresis in the critical conditions enabled, for the first time, the separation of hydrophilic polyacrylates according to their topology (branching) and their chain ends. This will allow meaningful and accurate characterization of their branched topologies as well as molar masses and progress in for advanced applications such as drug delivery or flocculation.

Assessing the quantification of acetylation in konjac glucomannan via ATR-FTIR and solid-state NMR spectroscopy.

Dietary fiber like konjac glucomannan (KGM) is important in maintaining good human health. There is no established method for quantifying the average degree of acetylation DA of this polysaccharide. Polysaccharides are notoriously difficult to dissolve. In this study, KGM could not be fully dissolved in common solvents and was characterized in the solid state. ATR-FTIR spectroscopy enabled a fast qualitative assessment of acetylation, selective to the outer layer of KGM particles, and identifying excipients like magnesium stearate. Average DA was quantified for the first time with solid-state 13C NMR in KGM: semi-quantitative measurements on the same arbitrary scale by cross polarization (1 to 2 days) were calibrated with a few longer single-pulse excitation measurements (approximately 1 week). DA values ranged from 4 to 8% of the hexoses in the backbone, in agreement with previously reported values. This method could be used for quality control and standardization of KGM products.

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.

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

Branched 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.

Engineering the Distinct Structure Interface of Subnano-alumina Domains on Silica for Acidic Amorphous Silica-Alumina toward Biorefining.

Amorphous silica-aluminas (ASAs) are important solid catalysts and supports for many industrially essential and sustainable processes, such as hydrocarbon transformation and biorefining. However, the wide distribution of acid strength on ASAs often results in undesired side reactions, lowering the product selectivity. Here we developed a strategy for the synthesis of a unique class of ASAs with unvarying strength of Brønsted acid sites (BAS) and Lewis acid sites (LAS) using double-flame-spray pyrolysis. Structural characterization using high-resolution transmission electron microscopy (TEM) and solid-state nuclear magnetic resonance (NMR) spectroscopy showed that the uniform acidity is due to a distinct nanostructure, characterized by a uniform interface of silica-alumina and homogeneously dispersed alumina domains. The BAS population density of as-prepared ASAs is up to 6 times higher than that obtained by classical methods. The BAS/LAS ratio, as well as the population densities of BAS and LAS of these ASAs, could be tuned in a broad range. In cyclohexanol dehydration, the uniform Brønsted acid strength provides a high selectivity to cyclohexene and a nearly linear correlation between acid site densities and cyclohexanol conversion. Moreover, the concerted action of these BAS and LAS leads to an excellent bifunctional Brønsted-Lewis acid catalyst for glucose dehydration, affording a superior 5-hydroxymethylfurfural yield.

Viscosimetric detection in size-exclusion chromatography (SEC/GPC): The Goldwasser method and beyond.

Size-exclusion chromatography (SEC or GPC) is the most widely used separation method to characterize polymers. The high level of complexity of most polymeric materials necessitates the use of not only concentration-sensitive detection but also structure-sensitive detection. Viscometry is usually used in conjunction with a concentration-sensitive detector and universal calibration to determine molecular weights of polymers. Goldwasser proposed to use a viscometer as a single detector to determine number-average molecular weights, M(n) (ACS Symposium Series, 521, 143). The method is particularly of interest when concentration-sensitive detection is not available, because the sample is isorefractive or not UV-absorbing, or because composition is not constant (copolymers). It has known very little applications so far. It actually does not only allow determining M(n), but also the number hydrodynamic volume distribution. This opens a wider range of applications for the Goldwasser method. Size-exclusion chromatography only yields inaccurate molecular weight distributions for some complex branched polymers. Hydrodynamic volume distributions have then a strong potential for comparative studies owing to their far higher accuracy. Our experimental tests highlight the fact that the method is highly sensitive to noise and careful optimization of the injection concentration is needed, but number distribution can be obtained as well as M(n).

Kinetics of in vitro digestion of starches monitored by time-resolved (1)H Nuclear Magnetic Resonance.

A (1)H NMR method is presented that monitors the initial and later stages of in vitro enzymatic digestion of starch suspensions. It allows, for the first time to our knowledge, the accurate analysis of the initial 5% of the extent of hydrolysis. This is significant because rapidly digested starch produces glucose that determines the blood glucose concentration immediately after ingestion of food. The two key hydrolytic enzymes, alpha-amylase and amyloglucosidase, showed clear systematic deviation from Michaelis-Menten kinetics as the starch or wheat flour substrate that was used changed its character during the reaction. Estimates of Michaelis-Menten parameters for amyloglucosidase and alpha-amylase were successfully found by analyzing two stages of digestion separately. The Michaelis-Menten constants for purified starch were (6.4 +/- 0.8) and (1.1 +/- 0.3) g dL(-1) (% w/v), respectively; and the maximum velocities of glucose release by amyloglucosidase, and short oligoglucosides and glucose by alpha-amylase were (1.9 +/- 0.4) x 10(-2) and (1.6 +/- 0.2) x 10(-2) mmol L(-1) s(-1) for the first stage of digestion, and (9.0 +/- 1.0) x 10(-3) and (4.7 +/- 1.4) x 10(-3) mmol L(-1) s(-1) for the second stage, giving a ratio of the two V(max) values of 2.1 and 3.4, respectively.

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.

Separation of complex branched polymers by size-exclusion chromatography probed with multiple detection.

Size-exclusion chromatography (SEC) separates polymers by hydrodynamic volume (the universal calibration principle). Molecular weights can be determined using viscometry (relying on universal calibration) and light scattering (independent of universal calibration). In the case of complex branched polyacrylates with tetrahydrofuran as eluent, universal calibration is valid, although the separation in term of molecular weight is incomplete: a given elution slice contains a range of molecular weights, described in terms of a 'local polydispersity'. The local polydispersity index decreases when the number of branches per chain increases and complete separation is reached for highly branched chains.

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.

Characterization of oligo(acrylic acid)s and their block co-oligomers.

Oligo(acrylic acid), oligoAA are important species currently used industrially in the stabilization of paints and also for the production of self-assembled polymer structures which have been shown to have useful applications in analytical separation methods and potentially in drug delivery systems. To properly tailor the synthesis of oligoAA, and its block co-oligomers synthesized by Reversible-Addition Fragmentation chain Transfer (RAFT) polymerization to applications, detailed knowledge about the chemical structure is needed. Commonly used techniques such as Size Exclusion Chromatography (SEC) and Electrospray Ionization-Mass Spectrometry (ESI-MS) suffer from poor resolution and non-quantitative distributions, respectively. In this work free solution Capillary Electrophoresis (CE) has been thoroughly investigated as an alternative, allowing for the separation of oligoAA by molar mass and the RAFT agent end group. The method was then extended to block co-oligomers of acrylic acid and styrene. Peak capacities up to 426 were observed for these 1D CE separations, 10 times greater than what has been achieved for Liquid Chromatography (LC) of oligostyrenes. To provide a comprehensive insight into the chemical structure of these materials 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used to provide an accurate average chain length and reveal the presence of branching. The chain length at which branching is detected was investigated with the results showing a degree of branching of 1% of the monomer units in oligoAA with an average chain length of 9 monomer units, which was the shortest chain length at which branching could be detected. This branching is suspected to be a result of both intermolecular and intramolecular transfer reactions. The combination of free solution CE and NMR spectroscopy is shown to provide a near complete elucidation of the chemical structure of oligoAA including the average chain length and branching as well as the chain length and RAFT agent end group distribution. Furthermore, the purity in terms of the dead chains and unreacted RAFT agent was quantified. The use of free solution CE and 1H NMR spectroscopy demonstrated in this work can be routinely applied to oligoelectrolytes and their block co-oligomers to provide an accurate characterization which allows for better design of the materials produced from these oligomers.

Biohybrid cellulose fibers: Toward paper materials with wet strength properties.

Herein, we report on the preparation of novel cellulose-PEG biohybrid papers with wet strength properties. The biohybrid paper sheets are obtained using a two-step procedure where ω- or α, ω-azide functionalized PEG chains are anchored onto alkyne-functionalized wood fibers through CuAAC ligation in mild and aqueous conditions. The incorporation of the PEG grafts mostly occurs at the periphery of the cellulose fibers and degrees of substitution up to 0.028 are obtained. The presence of PEG grafts significantly increases the tensile, burst and tear strength properties in the wet state, the reinforcement being more pronounced for fibers grafted with α,ω-azide PEG. This reinforcement is consistent with a relatively sparse hetero-crosslink reaction creating inter-fiber covalent bonds and forming a cellulose network within the cell wall.