Molar mass characterization of cationic methyl methacrylate-ethyl acrylate copolymers using size-exclusion chromatography with online multi-angle light scattering and refractometric detection

Size-exclusion chromatography (SEC) combined with online multi-angle light scattering (MALS) and refractometric (RI) detection has been employed for the molar mass characterisation of water-insoluble cationic methyl methacrylate-ethyl acrylate copolymers (Eudragit RS and RL). Due to their positive charge, cationic polymers are particularly difficult to separate on a SEC column, in worst cases being completely adsorbed on the oppositely charged packing material. This work has examined how a careful addition of salt (LiCl) to the copolymer solution in ethanol decreases the electrostatic interactions, clearly seen as a decrease in elution volume from the SEC column as well as an improved recovery. At a certain level of ionic strength, typically about 50 mM, the copolymer recovery from the SEC column reached 100% and molar mass distributions corresponding to the complete sample could be obtained. The combined MALS/RI detection gives the opportunity to measure the absolute molar mass independent of recovery and retention. Thus, in this study, it turned out to be a favourable tool for tracing the changes in elution behaviour of the charged copolymer as the ionic strength was increased.     

Micelle-sequestered dissociation of cationic DNA-intercalated drugs: unexpected surfactant-induced rate enhancement

Detergent sequestration using micelles as a hydrophobic sink for dissociated drug molecules is an established technique for determination of dissociation rates. The anionic surfactant molecules are generally assumed not to interact with the anionic DNA and thereby not to affect the rate of dissociation. By contrast, we here demonstrate that the surfactant molecules sodium dodecyl sulfate (SDS), sodium decyl sulfate, and sodium octyl sulfate all induce substantial rate enhancements of the dissociation of intercalators from DNA. Four different cationic DNA intercalators are studied with respect to surfactant-induced dissociation. Except for the smallest intercalator, ethidium, the dissociation rate constants increase monotonically with surfactant concentration both below cmc and (more strongly) above cmc, much more than expected from electrostatic effects of increased counterion concentration. The rate enhancement, most pronounced for the bulky, multicationic, hydrophobic DNA ligands in this study, indicates a reduction of the activation energy for the ligand to pass out from a deeply penetrating intercalation site of DNA. The discovery that surfactants enhance the rate of dissociation of cationic DNA-intercalators implies that rate constants previously determined by micelle-sequestered dissociation may have been overestimated. As an alternative, more reliable method, we suggest instead the addition of excess of dummy DNA as an absorbent for dissociated ligand.     

A new mechanism of benzophenone photoreduction in photoinitiated crosslinking of polyethylene and its model compounds

The photolytic products and a new photoreduction mechanism of benzophenone (BP) as a photoinitiator in the photocrosslinking of polyethylene (PE) and its model compounds (MD) have been studied by means of fluorescence, ESR, ¹³C and ¹H NMR spectroscopy. The fluorescence spectra from the PE and MD systems demonstrate that the main photoreduction product of BP (PPB) is benzpinacol formed by the recombination of two diphenylhydroxymethyl (K^•) radical intermediates. The ESR spectrum obtained from the UV irradiation of the MD/BP system gives positive evidence of K^• radicals. Two new PPB products: an isomer of benzpinacol with quinoid structure, 1‐phenyl‐hydroxymethylene‐4‐diphenyl‐hydroxymethyl‐2,5‐cyclohexa‐diene and three kinds of α‐alkylbenzhydrols have been detected and identified for the first time by ¹³C and ¹H NMR spectroscopy from the MD systems. The latter could be formed by the reactions of K^• radicals with alkyl radicals produced by hydrogen abstraction of the excited triplet state ³(BP)* from polyethylene or its model compounds. These results provide new experimental evidence for elucidating the photoreduction mechanism of BP in the photoinitiated crosslinking of polyethylene. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 999–1005, 2000     

Synthesis of 11C-amides using [11C]carbon monoxide and in situ activated amines by palladium-mediated carboxaminations

[11C]Carbon monoxide at low concentrations, aryl halides and amines were used in the palladium-mediated synthesis of twenty 11C-amides. In the study several approaches to improve the radiochemical yield were explored. Eight of the selected amides were prepared by in situ activation of the amines using lithium bis(trimethylsilyl)amide and the radiochemical yields of these reactions were improved compared to utilising a previous reported method. In the synthesis of 1-[carbonyl-11C]benzoyl-3-methyl-1H-indole (11) from 3-methyl-1H-indole (25), the corresponding organotin-amine was prepared prior to the acylation reaction. In a typical experiment, N-(4-hydroxyphenyl)[carbonyl-11C]acetamide (5) was prepared in 15% radiochemical yield using 4-aminophenol (20) but the yield increased to 63% when the amine was activated by lithium bis(trimethylsilyl)amide.     

Molecular mass distribution analysis of ethyl(hydroxyethyl)cellulose by size-exclusion chromatography with dual light-scattering and refractometric detection

Dual low-angle light scattering and refractometric detection coupled to size-exclusion chromatography provided proof for the presence of a low amount of stable aggregates/particles in ethyl(hydroxyethyl)cellulose. Unlike the correct size-exclusion chromatographic behavior of the parent polysaccharide itself, the aggregates exhibit variable size-dependent weak retention as a function of flow-rate and of ionic strength of the aqueous mobile phase. Therefore, determination of the molecular mass of non-aggregated polymer is possible in aqueous mobile phase containing 0.1 M NaCl under conditions at which aggregates are completely adsorbed on the column packing irrespective of the flow-rate used. Flow-rate and ionic strength-dependent variations of aggregate behavior as well as model size-exclusion experiments with latex particles indicate that they partly carry a minute charge and have a compact structure. Their weak retention under the separation conditions used suggests a difference in their surface chemistry when compared with the dissolved polymer coils which exhibit a correct size-exclusion behavior.     

Analysis of chiral amino acids in cerebrospinal fluid samples linked to different stages of Alzheimer disease

Chiral micellar electrokinetic chromatography with laser-induced fluorescence detection (chiral-MEKC-LIF) was used to investigate D- and L-amino acid contents in cerebrospinal fluid (CSF) samples related to different Alzheimer disease (AD) stages. CSF samples were taken from (i) control subjects (S1 pool), (ii) subjects showing a mild cognitive impairment who remained stable (S2 pool), (iii) subjects showing an mild cognitive impairment that progressed to AD (S3 pool) and (iv) subjects diagnosed with AD (S4 pool). The optimized procedure only needed 10 μL of CSF and it included sample cleaning, derivatization with FITC and chiral-MEKC-LIF separation. Eighteen standard amino acids were baseline separated with efficiencies up to 703,000 plates/m, high sensitivity (LODs in the nM range) and good resolution (values ranging from 2.6 to 9.5). Using this method, L-Arg, L-Leu, L-Gln, γ-aminobutyric acid, L-Ser, D-Ser, L-Ala, Gly, L-Lys, L-Glu and L-Asp were detected in all the CSF samples. S3 and S4 samples (i.e. AD subjects) showed significant lower amounts of L-Arg L-Lys, L-Glu and L-Asp compared to the non-AD S1 and S2 samples, showing in the S4 group the lowest amounts of L-Arg L-Lys, L-Glu and L-Asp. Moreover, γ-aminobutyric acid was significantly higher in AD subjects with the highest amount also found for S4. No significant differences were observed for the rest of amino acids including D-Ser. Based on the obtained chiral-MEKC-LIF data, it was possible to correctly classify all the samples into the four groups. These results demonstrate that the use of enantioselective procedures as the one developed in this work can provide some new light on the investigations of AD, including the discovery of new biomarkers related to different stages of AD.     

A multifaceted approach to hydrogen storage

The widespread adoption of hydrogen as an energy carrier could bring significant benefits, but only if a number of currently intractable problems can be overcome. Not the least of these is the problem of storage, particularly when aimed at use onboard light-vehicles. The aim of this overview is to look in depth at a number of areas linked by the recently concluded HYDROGEN research network, representing an intentionally multi-faceted selection with the goal of advancing the field on a number of fronts simultaneously. For the general reader we provide a concise outline of the main approaches to storing hydrogen before moving on to detailed reviews of recent research in the solid chemical storage of hydrogen, and so provide an entry point for the interested reader on these diverse topics. The subjects covered include: the mechanisms of Ti catalysis in alanates; the kinetics of the borohydrides and the resulting limitations; novel transition metal catalysts for use with complex hydrides; less common borohydrides; protic-hydridic stores; metal ammines and novel approaches to nano-confined metal hydrides.     

Isolation of Escherichia coli inner membranes by metal affinity two-phase partitioning

As reduction of sample complexity is a central issue in membrane proteomic research, the need for new pre-fractionation methods is significant. Here we present a method for fast and efficient enrichment of Escherichia coli inner membranes expressing a His-tagged integral membrane L-fucose-proton symporter (FucP). An enriched inner membrane fraction was obtained from a crude membrane mixture using affinity two-phase partitioning in combination with nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads. Due to interaction between the beads and FucP, inner membranes were selectively partitioned to the bottom phase of a polymer/polymer aqueous two-phase system consisting of poly(ethylene glycol) (PEG) and dextran. The partitioning of membranes was monitored by assaying the activity of an inner membrane marker protein and measuring the total protein content in both phases. The enrichment of inner membrane proteins in the dextran phase was also investigated by proteomic methodology, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), trypsin digestion and liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS). Using a high level of significance (99.95%) in the subsequent database search, 36 proteins assigned to the inner membrane were identified in the bottom phase, compared to 29 when using the standard sucrose gradient centrifugation method for inner membrane isolation. Furthermore, metal affinity two-phase partitioning was up to 10 times faster than sucrose gradient centrifugation. The separation conditions in these model experiments provide a basis for the selective isolation of E. coli membranes expressing His-tagged proteins and can therefore facilitate research on such membrane proteomes.     

Influence of nanotopography on phospholipid bilayer formation on silicon dioxide

We have investigated the effect of well-defined nanoscale topography on the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicle adsorption and supported phospholipid bilayer (SPB) formation on SiO2 surfaces using a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). Unilamellar lipid vesicles with two different sizes, 30 and 100 nm, were adsorbed on pitted surfaces with two different pit diameters, 110 and 190 nm, as produced by colloidal lithography, and the behavior was compared to results obtained on flat surfaces. In all cases, complete bilayer formation was observed after a critical coverage of adsorbed vesicles had been reached. However, the kinetics of the vesicle-to-bilayer transformation, including the critical coverage, was significantly altered by surface topography for both vesicle sizes. Surface topography hampered the overall bilayer formation kinetics for the smaller vesicles, but promoted SPB formation for the larger vesicles. Depending on vesicle size, we propose two modifications of the precursor-mediated vesicle-to-bilayer transformation mechanism used to describe supported lipid bilayer formation on the corresponding flat surface. Our results may have important implications for various lipid-membrane-based applications using rough or topographically structured surfaces.