New Developments in Multidimensional Chromatography of Complex Polymers

Summary: Complex polymers are distributed in more than one direction of molecular heterogeneity. In addition to the molar mass distribution, they are frequently distributed with respect to chemical composition, functionality, and molecular heterogeneity. One approach for the analysis of the heterogeneity of complex polymers is their chromatographic separation by combining different separation mechanisms. A typical experimental protocol includes the separation of the sample according to composition to yield fractions that are chemically homogeneous. These fractions are transferred to a size‐selective separation method and analyzed with respect to molar mass. As a result of this two‐dimensional (2D) separation, information on both types of molecular heterogeneity is obtained. So far, 2D chromatography has been applied mostly to polymers that are soluble in organic solvents. There are several problems related to the use of aqueous mobile phases in polymer chromatography. These problems relate to the very polar or ionic character of the polymers and the experimental conditions, including the use of salt‐containing eluents. The present paper addresses the different parameters that influence the chromatographic experiments. For a model polymer system resulting from the grafting of methacrylic acid (MAA) onto poly(ethylene glycol) (PEG), i.e., PEG‐g‐PMAA, it will be shown that different chromatographic techniques including SEC, LC‐CC, and 2D chromatography, as well as coupled LC‐CC/FT‐IR can be used to analyze the molecular complexity of the copolymers.

LC‐CC/FT‐IR spectra of a PEG‐g‐PMAA sample as function of the elution volume.     

Influence of substrate and product concentrations on the production of cyclodextrins by CGTase of Bacillus firmus,strain no. 37

The influence of substrate or product level on the initial velocity of cyclodextrin (CD) production by cyclodextringlycosyltransferase from a Brazilian isolate of Bacillus firmus was studied. Our results indicate that the product γ-CD is a stronger inhibitor to the reaction than β-CD. Small saccharides could also inhibit CD production, although to a lesser extent than the products, and maltose was the strongest inhibitor among small saccharides. Increasing substrate concentration resulted in greater reduction on enzyme activity for the formation of β-CD than for γ-CD. We modeled the kinetics of CD production with a set of four reversible reactions including the cyclization/coupling reaction that forms/opens CDs, and three disproportionation reactions. Our model on the initial velocity data explained well the substrate inhibition phenomenon. Kinetic parameters were determined by fitting the initial velocity data into our model.     

Ceramic Foams from a Preceramic Polymer and Polyurethanes: Preparation and Morphological Investigations

Open-cell ceramic foams were obtained from a preceramic polymer (silicone resin) and blown polyurethanes. The preceramic polymer, which is crosslinked by condensation of silanol groups, was dissolved in CH2Cl2 and added to a liquid polyol containing the surfactant and the amine catalyst. Isocyanate was then added to the mixture and the foam was obtained through a twofold blowing mechanism (physical and chemical blowing). The morphology of the expanded polyurethane, which can be flexible or semirigid, characterized the final structure of the ceramic foam. The materials obtained were pyrolyzed in a nitrogen flux at temperatures of 1000–1200°C, thus allowing for the polymer-to-ceramic transformation to occur in the preceramic polymer. The ceramic foams produced in this way consisted of an amorphous silicon oxycarbide ceramic (SiOC). They presented a density ranging from 0.1 to 0.3 g/cm3. The average pore diameter ranged from 200 to 400 m and they possessed 80 to 90% open porosity.     

Shell-cross-linked vesicles synthesized from block copolymers of Poly(D,L-lactide) and Poly(N-isopropyl acrylamide) as thermoresponsive nanocontainers

A polylactide (D,L-PLA) macroRAFT agent was prepared by utilizing a hydroxyl-functional trithiocarbonate as a coinitiator for the ring-opening polymerization. The length of the resultant polymer was controlled by the concentration of the coinitiator leading to the formation of two PLA polymers with M(n) = 12500 g mol(-)(1) (PDI = 1.46) and M(n) = 20500 g mol(-)(1) (PDI = 1.38) each with omega-trithiocarbonate functionality. Chain extension of PLA via the RAFT (free radical) polymerization of N-isopropyl acrylamide (NIPAAm) resulted in the formation of amphiphilic block copolymers with the PNIPAAm block increasing in size with conversion. TEM measurements of the aggregates obtained by self-organization of the block copolymers in aqueous solutions indicated the formation of vesicles. The sizes of these aggregates were influenced by the ratio of both blocks and the molecular weight of each block. The lower critical solution temperature (LCST) of the block copolymer was largely unaffected by the size of each block. UV turbidity measurements indicated a higher LCST for the block copolymers than for the corresponding PNIPAAm homopolymers. Stabilization of the vesicles was attained by a cross-linking chain extension of the PNIPAAm block using hexamethylene diacrylate. As the trithiocarbonate group was located between the PLA and PNIPAAm blocks, the chain extension resulted in a cross-linked layer between the core and corona of the vesicles.     

A new tripodal receptor for molecular recognition of monosaccharides. A paradigm for assessing glycoside binding affinities and selectivities by 1H NMR spectroscopy

A new tripodal receptor for the recognition of monosaccharides is described. The prototypical host 1 features a 1,3,5-substituted 2,4,6-triethylbenzene scaffold bearing three convergent H-bonding units. The binding ability of the t-octyl derivative 1a toward a set of octylglycosides of biologically relevant monosaccharides, including Glc, Gal, Man, and GlcNAc, was investigated by 1H NMR in CDCl3. A protocol for the correct evaluation of binding affinities was established, which can be generally applied for the recognition of monosaccharides by 1H NMR spectroscopy. A three-constant equilibrium model, including 1:1 and 2:1 host-guest association and dimerization of the receptor, was ascertained for the interaction of 1a with all the investigated glycosides. An affinity index, which we defined median binding concentration BC50 in analogy to the IC50 parameter, intended to address the general issue of comparing dimensionally heterogeneous binding data, and a limiting BC0(50)quantity describing intrinsic binding affinities were developed for evaluating the results. BC0(50) values for 1a range from 1 to 6 mM, indicating an intrinsic binding affinity in the millimolar range and a selectivity factor of 5 toward the investigated glycosides. The treatment has been extended to include any generic host-guest system involved in single or multiple binding equilibria.     

Production and purification of CGTase of alkalophylicBacillus isolated from Brazilian soil

Alkalophylic bacilli that produce cyclodextringlycosyltransferase (CGTase) were isolated from Brazilian soil, with a scheme of two plating steps. In the first step, the bacterial isolate forms a halo in the cultivation medium that contains γ-cyclodextrin (CD) complexing dyes. The CGTase of an isolate was purified 157-fold by biospecific affinity chromatography, with β-CD showing a mol wt of 77,580 Daltons. It produces a γ- to β-CD ratio of 0.156 and a small amount of α-CD, using maltodextrin 10% as substrate, at 50°C, pH 8.0 and 22 h reaction time, reaching 21.4% conversion of the substrate to cyclodextrins. In the second screening step, the isolates chosen give larger halos with β-CD complexing dyes, and smaller halos with β-CD complexing dyes, leading to a 30% improvement in γ-CD selectivity, although at lower total yield for cyclodextrins (11.5%).     

Carbon and nitrogen isotope analysis of atrazine and desethylatrazine at sub-microgram per liter concentrations in groundwater

Environmental degradation of organic micropollutants is difficult to monitor due to their diffuse and ubiquitous input. Current approaches—concentration measurements over time, or daughter-to-parent compound ratios—may fall short, because they do not consider dilution, compound-specific sorption characteristics or alternative degradation pathways. Compound-specific isotope analysis (CSIA) offers an alternative approach based on evidence from isotope values. Until now, however, the relatively high limits for precise isotope analysis by gas chromatography—isotope ratio mass spectrometry (GC-IRMS) have impeded CSIA of sub-microgram-per-liter scale micropollutant concentrations in field samples. This study presents the first measurements of C and N isotope ratios of the herbicide atrazine and its metabolite desethylatrazine at concentrations of 100 to 1,000 ng/L in natural groundwater samples. Solid-phase extraction and preparative HPLC were tested and validated for preconcentration and cleanup of groundwater samples of up to 10 L without bias by isotope effects. Matrix interferences after solid-phase extraction could be greatly reduced by a preparative HPLC cleanup step prior to GC-IRMS analysis. Sensitivity was increased by a factor of 6 to 8 by changing the injection method from large-volume to cold-on-column injection on the GC-IRMS system. Carbon and nitrogen isotope values of field samples showed no obvious correlation with concentrations or desethylatrazine-to-atrazine ratios. Contrary to expectations, however, δ ¹³ C values of desethylatrazine were consistently less negative than those of atrazine from the same sites. Potentially, this line of evidence may contain information about further desethylatrazine degradation. In such a case, the common practice of using desethylatrazine-to-atrazine ratios would underestimate natural atrazine degradation.     

Quercetin-PVP K25 solid dispersions

Quercetin is a flavonoid very well studied and has already entered clinical trials emerging as prospective anticancer drug candidate. In addition, quercetin has being reported to its free-radical scavenging activity and suggests potential uses for the prevention and treatment of pathologies as atherosclerosis, chronic inflammation, and others. However, quercetin is sparingly soluble in water, which may be responsible for its limited absorption upon oral administration. The solid dispersion of quercetin with polyvinylpyrrolidone Kollidon^® 25 (PVP K25) suggests an interesting way to increase quercetin solubility, antioxidant activity, and consequently bioavailability. Then, the purpose of this study was to prepare solid dispersions of quercetin with PVP K25 and evaluate their thermal characterization, antioxidant activity and quercetin improvement solubility. For this purpose, quercetin-PVP K25 solutions were dried and quercetin-PVP K25 solids were obtained. The formation of quercetin-PVP K25 solid dispersion was evaluated by solubility studies, powder X-ray diffraction (XRD), fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), thermogravimetry (TG), and antioxidant activity. It was observed that PVP K25 was able to provide quercetin clear aqueous solutions and that quercetin solubility was increased in a PVP K25 concentration dependent manner, improving solubility even 436-fold the pure quercetin. The results obtained with XRD, FT-IR, DSC, and TG demonstrated possible quercetin-PVP K25 solid dispersion formation. Besides, the antioxidant activity of the quercetin-PVP K25 solid dispersions dissolved in aqueous solution and pure quercetin dissolved in methanol showed IC₅₀ value of 0.61 ± 0.03 and 1.00 ± 0.02 μg/mL, respectively, demonstrating that the solid dispersions presented a significant increase in antioxidant activity (P < 0.05). Putting results together, it was possible to conclude there was the formation of quercetin-PVP K25 solid dispersion.     

Electrochemical Assay to Quantify the Hydroxyl Radical Scavenging Activity of Medicinal Plant Extracts

A new electrochemical assay has been developed which is based on quantifying the attack of OH^. radicals on a self‐assembled thiol monolayer on electrodes in the presence and absence of antioxidants. The OH^. radicals were produced in Fenton solutions. The radical scavengers diminish the concentration of the OH^. radicals in the Fenton solution, and the extent of thiol layer destruction is monitored with the help of a redox probe in solution, the signal of which is only generated at SAM‐free electrode surface. The results of the electrochemical assay were compared with those of the standard DPPH^. assay. In case of each plant extract, an excellent linear correlation was observed between the radical scavenging activity and the concentration of plant extracts; however, the scavenging properties of different plants strongly differ in the electrochemical and the DPPH^. assay. Since the proposed new method probes the reactivity of radical scavengers towards OH^., one of the most important reactive oxygen radicals in tissues, it is supposed to provide much more relevant information than the DPPH^. or other assays.