Revealing the Phenotypic and Genomic Background for PHA Production from Rapeseed-Biodiesel Crude Glycerol Using Photobacterium ganghwense C2.2.

Polyhydroxyalkanoates (PHA) are promising biodegradable and biocompatible bioplastics, and extensive knowledge of the employed bacterial strain's metabolic capabilities is necessary in choosing economically feasible production conditions. This study aimed to create an in-depth view of the utilization of Photobacterium ganghwense C2.2 for PHA production by linking a wide array of characterization methods: metabolic pathway annotation from the strain's complete genome, high-throughput phenotypic tests, and biomass analyses through plate-based assays and flask and bioreactor cultivations. We confirmed, in PHA production conditions, urea catabolization, fatty acid degradation and synthesis, and high pH variation and osmotic stress tolerance. With urea as a nitrogen source, pure and rapeseed-biodiesel crude glycerol were analyzed comparatively as carbon sources for fermentation at 20 °C. Flask cultivations yielded 2.2 g/L and 2 g/L PHA at 120 h, respectively, with molecular weights of 428,629 g/mol and 81,515 g/mol. Bioreactor batch cultivation doubled biomass accumulation (10 g/L and 13.2 g/L) in 48 h, with a PHA productivity of 0.133 g/(L·h) and 0.05 g/(L·h). Thus, phenotypic and genomic analyses determined the successful use of Photobacterium ganghwense C2.2 for PHA production using urea and crude glycerol and 20 g/L NaCl, without pH adjustment, providing the basis for a viable fermentation process.

High natural PHA production from acetate in Cobetia sp. MC34 and Cobetia marina DSM 4741T and in silico analyses of the genus specific PhaC2 polymerase variant.

BACKGROUND: Several members of the bacterial Halomonadacea family are natural producers of polyhydroxyalkanoates (PHA), which are promising materials for use as biodegradable bioplastics. Type-strain species of Cobetia are designated PHA positive, and recent studies have demonstrated relatively high PHA production for a few strains within this genus. Industrially relevant PHA producers may therefore be present among uncharacterized or less explored members. In this study, we characterized PHA production in two marine Cobetia strains. We further analyzed their genomes to elucidate pha genes and metabolic pathways which may facilitate future optimization of PHA production in these strains. RESULTS: Cobetia sp. MC34 and Cobetia marina DSM 4741T were mesophilic, halotolerant, and produced PHA from four pure substrates. Sodium acetate with- and without co-supplementation of sodium valerate resulted in high PHA production titers, with production of up to 2.5 g poly(3-hydroxybutyrate) (PHB)/L and 2.1 g poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/L in Cobetia sp. MC34, while C. marina DSM 4741T produced 2.4 g PHB/L and 3.7 g PHBV/L. Cobetia marina DSM 4741T also showed production of 2.5 g PHB/L from glycerol. The genome of Cobetia sp. MC34 was sequenced and phylogenetic analyses revealed closest relationship to Cobetia amphilecti. PHA biosynthesis genes were located at separate loci similar to the arrangement in other Halomonadacea. Further genome analyses revealed some differences in acetate- and propanoate metabolism genes between the two strains. Interestingly, only a single PHA polymerase gene (phaC2) was found in Cobetia sp. MC34, in contrast to two copies (phaC1 and phaC2) in C. marina DSM 4741T. In silico analyses based on phaC genes show that the PhaC2 variant is conserved in Cobetia and contains an extended C-terminus with a high isoelectric point and putative DNA-binding domains. CONCLUSIONS: Cobetia sp. MC34 and C. marina DSM 4741T are natural producers of PHB and PHBV from industrially relevant pure substrates including acetate. However, further scale up, optimization of growth conditions, or use of metabolic engineering is required to obtain industrially relevant PHA production titers. The putative role of the Cobetia PhaC2 variant in DNA-binding and the potential implications remains to be addressed by in vitro- or in vivo methods.

Studies of Emission Processes of Polymer Additives into Water Using Quartz Crystal Microbalance-A Case Study on Organophosphate Esters.

Plastic materials contain various additives, which can be released during the entire lifespan of plastics and pose a threat to the environment and human health. Despite our knowledge on leakage of additives from products, accurate and rapid approaches to study emission kinetics are largely lacking, in particular, methodologies that can provide in-depth understanding of polymer/additive interactions. Here, we report on a novel approach using quartz crystal microbalance (QCM) to measure emissions of additives to water from polymer films spin-coated on quartz crystals. The methodology, being accurate and reproducible with a standard error of ±2.4%, was applied to a range of organophosphate esters (OPEs) and polymers with varying physicochemical properties. The release of most OPEs reached an apparent steady-state within 10 h. The release curves for the studied OPEs could be fitted using a Weibull model, which shows that the release is a two-phase process with an initial fast phase driven by partitioning of OPEs readily available at or close to the polymer film surface, and a slower phase dominated by diffusion in the polymer. The kinetics of the first emission phase was mainly correlated with the hydrophobicity of the OPEs, whereas the diffusion phase was weakly correlated with molecular size. The developed QCM-based method for assessing and studying release of organic chemicals from a polymeric matrix is well suited for rapid screening of additives in efforts to identify more sustainable replacement polymer additives with lower emission potential.

Selective Enrichment of Phosphorylated Peptides by Monolithic Polymers Surface Imprinted with bis-Imidazolium Moieties by UV-Initiated Cryopolymerization.

Reversible protein phosphorylation on serine, threonine, and tyrosine residues is essential for fast, specific, and accurate signal transduction in cells. Up to now, the identification and quantification of phosphorylated amino acids, peptides, and proteins continue to be one of the significant challenges in contemporary bioanalytical research. In this paper, a series of surface grafted monoliths in the capillary format targeting phosphorylated serine has been prepared by first synthesizing a monolithic core substrate material based on trimethylolpropane trimethacrylate, onto which a thin surface-imprinted layer was established by oriented photografting of a variety of mono- and bis-imidazolium host monomers at subzero temperature, using six different continuous or pulsed UV light sources. The imprinted monolith capillaries were evaluated in a capillary liquid chromatographic system connected to a mass spectrometer in order to test the specific retention of phosphorylated peptides. Site-specific recognition selectivity and specificity for phosphorylated serine was demonstrated when separating amino acids and peptides, proving that the optimized materials could be used as novel trapping media in affinity-based phosphoproteomic analysis.

Molecularly Imprinted Porous Monolithic Materials from Melamine-Formaldehyde for Selective Trapping of Phosphopeptides.

Thirty-five melamine-formaldehyde (MF) monolithic materials with bimodal pore distributions were synthesized in fused silica capillaries by catalyst-free polycondensation, starting with an aqueous MF precondensate, using acetonitrile as the macroporogen and a variety of aliphatic polyethers and triblock copolymeric surfactants as porogens and mesoporogens, respectively. By varying the prepolymer composition and the type and molecular weight of the polymeric porogen components, a library of porous monolithic materials was produced, covering a range of meso- and macroporous properties. A multivariate evaluation revealed that the amount of surfactant was the strongest contributor to specific surface area and pore volume and to the inversely related mesopore size, whereas the macropore dimensions were controlled mainly by the amount of aliphatic polyether porogen. One of these capillary monoliths, chosen based on the combination of meso- and macropores providing optimal percolative flow and accessible surface area, was synthesized in the presence of N-Fmoc and O-Et protected phosphoserine and phosphotyrosine to prepare molecularly imprinted monoliths with surface layers selective for phosphopeptides. These imprinted monoliths were characterized alongside nonimprinted monoliths by a variety of techniques and finally evaluated by liquid chromatography-mass spectrometry in the capillary format to assess their abilities to trap and release phosphorylated amino acids and peptides from partly aqueous media. Selective enrichment of phosphorylated targets was demonstrated, suggesting that these materials could be useful as trapping media in affinity-based phosphoproteomics.

Elucidation of the Binding Orientation in α2,3- and α2,6-Linked Neu5Ac-Gal Epitopes toward a Hydrophilic Molecularly Imprinted Monolith.

N-Acetylneuraminic acid and its α2,3/α2,6-glycosidic linkages with galactose (Neu5Ac-Gal) are major carbohydrate antigen epitopes expressed in various pathological processes, such as cancer, influenza, and SARS-CoV-2. We here report a strategy for the synthesis and binding investigation of molecularly imprinted polymers (MIPs) toward α2,3 and α2,6 conformations of Neu5Ac-Gal antigens. Hydrophilic imprinted monoliths were synthesized from melamine monomer in the presence of four different templates, namely, N-acetylneuraminic acid (Neu5Ac), N-acetylneuraminic acid methyl ester (Neu5Ac-M), 3'-sialyllactose (3SL), and 6'-sialyllactose (6SL), in a tertiary solvent mixture at temperatures varying from -20 to +80 °C. The MIPs prepared at cryotemperatures showed a preferential affinity for the α2,6 linkage sequence of 6SL, with an imprinting factor of 2.21, whereas the α2,3 linkage sequence of 3SL resulted in nonspecific binding to the polymer scaffold. The preferable affinity for the α2,6 conformation of Neu5Ac-Gal was evident also when challenged by a mixture of other mono- and disaccharides in an aqueous test mixture. The use of saturation transfer difference nuclear magnetic resonance (STD-NMR) on suspensions of crushed monoliths allowed for directional interactions between the α2,3/α2,6 linkage sequences on their corresponding MIPs to be revealed. The Neu5Ac epitope, containing acetyl and polyalcohol moieties, was the major contributor to the sequence recognition for Neu5Ac(α2,6)Gal(ß1,4)Glc, whereas contributions from the Gal and Glc segments were substantially lower.

Terminally Phosphorylated Triblock Polyethers Acting Both as Templates and Pore-Forming Agents for Surface Molecular Imprinting of Monoliths Targeting Phosphopeptides.

The novel process reported here described the manufacture of monolithic molecularly imprinted polymers (MIPs) using a terminally functionalized block copolymer as the imprinting template and pore-forming agent. The MIPs were prepared through a step-growth polymerization process using a melamine-formaldehyde precondensate in a biphasic solvent system. Despite having a relatively low imprinting factor, the use of MIP monolith in liquid chromatography demonstrated the ability to selectively target desired analytes. An MIP capillary column was able to separate monophosphorylated peptides from a tryptic digest of bovine serum albumin. Multivariate data analysis and modeling of the phosphorylated and nonphosphorylated peptide retention times revealed that the number of phosphorylations was the strongest retention contributor for peptide retention on the monolithic MIP capillary column.

Synthesis of poly(N-[tris(hydroxymethyl)methyl]acrylamide) functionalized porous silica for application in hydrophilic interaction chromatography.

Porous silica coated by a highly hydrophilic and nonionic tentacle-type polymeric layer was synthesized by free radical "grafting from" polymerization of N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-2-propenamide (TRIS-acrylamide) in partly aqueous solutions. The radical initiator sites were incorporated on the silica surfaces via a two-step reaction comprising thionyl chloride activation and subsequent reaction with tert-butyl hydroperoxide. The surface-bound tert-butylperoxy groups were then used as thermally triggered initiators for graft polymerization of TRIS-acrylamide. The synthesized materials were characterized by diffusive reflectance Fourier transform infrared specotroscopy, X-ray photoelectron spectroscopy, and CHN elemental analysis. Photon correlation spectroscopy was used to determine changes in ζ-potentials resulting from grafting, (29)Si magic angle spinning nuclear magnetic resonance spectroscopy (MAS-NMR) spectroscopy was used to assess the ratio of silanol to siloxane groups in the substrate and the grafted material, and the changes in surface area and mesopore distribution were determined by nitrogen cryosorption. Chromatographic evaluation in hydrophilic interaction chromatography (HILIC) mode showed that the materials were suitable for use as stationary phases, featuring good separation efficiency, a comparatively high retention, and a selectivity that differed from most commercially available HILIC phases. A comparison of this neutral phase with a previously reported N-(2-hydroxypropyl)-linked TRIS-type hydrophilic tentacle phase with weak anion exchange functionality revealed substantial differences in retention patterns.

Discrimination between sialic acid linkage modes using sialyllactose-imprinted polymers.

Glycosylation plays an important role in various pathological processes such as cancer. One key alteration in the glycosylation pattern correlated with cancer progression is an increased level as well as changes in the type of sialylation. Developing molecularly-imprinted polymers (MIPs) with high affinity for sialic acid able to distinguish different glycoforms such as sialic acid linkages is an important task which can help in early cancer diagnosis. Sialyllactose with α2,6' vs. α2,3' sialic acid linkage served as a model trisaccharide template. Boronate chemistry was employed in combination with a library of imidazolium-based monomers targeting the carboxylate group of sialic acid. The influence of counterions of the cationic monomers and template on their interactions was investigated by means of 1H NMR titration studies. The highest affinities were afforded using a combination of Br- and Na+ counterions of the monomers and template, respectively. The boronate ester formation was confirmed by MS and 1H/11B NMR, indicating 1 : 2 stoichiometries between sialyllactoses and boronic acid monomer. Polymers were synthesized in the form of microparticles using boronate and imidazolium monomers. This combinatorial approach afforded MIPs selective for the sialic acid linkages and compatible with an aqueous environment. The molecular recognition properties with respect to saccharide templates and glycosylated targets were reported.

Differences in porous characteristics of styrenic monoliths prepared by controlled thermal polymerization in molds of varying dimensions.

Nitroxide-mediated polymerization was used as a model system for preparing styrenic monolithic materials with significant mesopore contents in different mold formats, with the aim of assessing the validity of pore characterization of capillary monoliths by analysis of parallel bulk polymerized precursor solution. Capillary monoliths were prepared in 250 microm id fused silica tubes (quadruplicate samples, in total 17 m), and the batch polymerizations were carried out in parallel in 100 microL microvials and regular 2 mL glass vials, both in quintuplicate. The monoliths recovered from the molds were characterized for their meso- and macroporous properties by nitrogen sorptiometry (three repeated runs on each sample), followed by a single analysis by mercury intrusion porosimetry. A total of 14 monolith samples were thus analyzed. A Grubbs' test identified one regular vial sample as an outlier in the sorptiometric surface area measurements, and data from this sample were consequently excluded from the pore size calculations, which are based on the same nitrogen sorption data, and also from the mercury intrusion data set. The remaining data were subjected to single factor analyses of variance analyses to test if the porous properties of the capillary monoliths were different from those of the bulk monoliths prepared in parallel. Significant differences were found between all three formats both in their meso- and macroporous properties. When the dimension was shrunk from conventional vial to capillary size, the specific surface area decreased from 52.2+/-4.7 to 34.6+/-1.7 m(2)/g. This decrease in specific surface area was accompanied by a significant shift in median diameter of the through-pores, from 310+/-3.9 to 544+/-13 nm. None of these differences were obvious from the scanning electron micrographs that were acquired for each sample type. The common practice of determining the mesopore characteristics from analysis of samples prepared by parallel bulk polymerization and looking for changes in the macropore structure by visual assessment of SEMs are therefore both rather questionable, at least for monoliths of the kind used in this study.

Tris(hydroxymethyl)aminomethane-functionalized silica particles and their application for hydrophilic interaction chromatography.

A new method is presented for synthesizing a highly hydrophilic silica-based material for use in hydrophilic interaction chromatography. Porous silica particles used as a starting substrate were modified with 3-bromopropyl trichlorosilane and grafted with glycidyl methacrylate by controlled ("living") atom transfer radical polymerization in order to introduce an oxirane-carrying reactive tentacle layer on the silica surface. The grafted material was thereafter subject to an oxirane ring opening reaction with tris(hydroxy-methyl)aminomethane in dimethylformamide to yield a polymer-bound equivalent of the well known and highly hydrophilic "TRIS" buffering substance. Chemical characterization was done by diffuse reflectance FT-IR, X-ray photoelectron spectroscopy, elemental analysis, and (1)H NMR. Porosity and surface area examination was done with Brunauer-Emmett-Teller. Chromatographic application of the material was evaluated by separations of nucleic bases, small organic acids, and common nucleotides under mixed hydrophilic interaction chromatography and weak anion exchange conditions.

Plasma brominated polymer particles as grafting substrate for thiol-terminated telomers.

A combined surface activation and "grafting to" strategy was developed to convert divinylbenzene particles into weak cation exchangers suitable for protein separation. The initial activation step was based on plasma modification with bromoform, which rendered the particles amenable to further reaction with nucleophiles by introducing Br to a surface content of 11.2 atom-%, as determined by X-ray photoelectron spectroscopy. Grafting of thiol-terminated glydicyl methacrylate telomers to freshly plasma activated surfaces was accomplished without the use of added initiator, and the grafting was verified both by reduction in bromine content and the appearance of sulfur-carbon linkages, showing that the surface grafts were covalently bonded. Following grafting the attached glydicyl methacrylate telomer tentacles were further modified by a two-step procedure involving hydrolysis to 2,3-hydroxypropyl groups and conversion of hydroxyl groups to carboxylate functionality by succinic anhydride. The final material was capable of baseline separating four model proteins in 3 min by gradient cation exchange chromatography in a fully aqueous eluent.

Probing the retention mechanism of small hydrophilic molecules in hydrophilic interaction chromatography using saturation transfer difference nuclear magnetic resonance spectroscopy.

The interactions and dynamic behavior of a select set of polar probe solutes have been investigated on three hydrophilic and polar commercial stationary phases using saturation transfer difference 1H nuclear magnetic resonance (STD-NMR) spectroscopy under magic angle spinning conditions. The stationary phases were equilibrated with a select set of polar solutes expected to show different interaction patterns in mixtures of deuterated acetonitrile and deuterium oxide, with ammonium acetate added to a total concentration that mimics typical eluent conditions for hydrophilic interaction chromatography (HILIC). The methylene groups of the stationary phases were selectively irradiated to saturate the ligand protons, at frequencies that minimized the overlaps with reporting protons in the test probes. During and after this radiation, the saturation rapidly spreads to all protons in the stationary phase by spin diffusion, and from those to probe protons in contact with the stationary phase. Probe protons that have been in close contact with the stationary phase and subsequently been released to the solution phase will have been more saturated due to a more efficient transfer of spin polarization by the nuclear Overhauser effect. They will therefore show a higher signal after processing of the data. Saturation transfers to protons in neutral and charged solutes could in some instances show clear orientation patterns of these solutes towards the stationary phases. The saturation profile of formamide and its N-methylated counterparts showed patterns that could be interpreted as oriented hydrogen bond interaction. From these studies, it is evident that the functional groups on the phase surface have a strong contribution to the selectivity in HILIC, and that the retention mechanism has a significant contribution from oriented interactions.

Grafting of silica with sulfobetaine polymers via aqueous reversible addition fragmentation chain transfer polymerization and its use as a stationary phase in HILIC.

Porous silica particles of 3 microm diameter and 100 A nominal pore size were first activated for vinylic polymerization by functionalization with 3-methacryloyloxypropyl trimethoxysilane (MAPTMS) and thereafter dressed with zwitterionic grafts of the sulfoalkylbetaine type in the "grafting through" fashion by polymerizing 3-(2-(N-methacryloyloxyethyl)-N,N-dimethylammonio)propane sulfonate (SPE), using either free radical polymerization or controlled reversible addition fragmentation chain transfer polymerization (RAFT). Particles polymerized using RAFT had a lower overall coating which seemed to be more evenly distributed in the pore volume. Both approaches resulted in columns with similar separation properties in HILIC mode.

Thermally induced dissolution/precipitation--a simple approach for the preparation of macroporous monoliths from linear aliphatic polyamides.

A versatile way of preparing macroporous monolithic materials from linear aliphatic polyamides is presented. Simply, polyamide pellets were treated in benzyl alcohol (BA) at elevated temperature, causing dissolution by interchain hydrogen bond disruption. Subsequent cooling below the upper critical solution temperature (UCST) resulted in precipitation and partial restoration of the semicrystalline polymer, which is organized into network structures. The final steps were a solvent exchange of BA for methanol, followed by drying to form monolithic entities. A number of polyamides ranging from hydrophilic to hydrophobic were tested and under the experimental conditions, poly(1-aza-2-cycloheptanone (PA6) and (poly-[imino-1,6-hexanediylimino{1,10-dioxo-1,10-decanediyl}] (PA610) yielded entities with macroporous properties that were deemed useful for liquid chromatography. The morphological features and porous properties of the monoliths produced by this dissolution-precipitation procedure were studied by scanning electron microscopy, adsorption/desorption of N(2)(g) according to the Brunauer-Emmett-Teller (BET) principle, and mercury intrusion porosimetry. Degradation of the polymer backbone was noticeable when the dissolution time was extended and shortening of the polymer chains was confirmed by MALDI-MS, viscosity measurements, X-ray photoelectron spectroscopy (XPS), and potentiometric titration. When the heating was limited to the time it took to dissolve the polymers, mechanically stable monoliths could be obtained. The dissolution/heat treatment time further seemed to be useful for controlling the macroporous morphology.

Functionalization of epoxy-based monoliths for ion exchange chromatography of proteins.

Macroporous epoxy-based monoliths prepared by emulsion polymerization have been modified for use in ion exchange chromatography (IEC) of proteins. Strong anion exchange functionality was established by iodomethane quaternization of tertiary amine present on the monolith surface as a part of the polymer backbone. The modification pathway to cation exchange materials was via incorporation of glycidyl methacrylate (GMA) brushes which were coated using atom transfer radical polymerization (ATRP). Strong (SO(3)(-)) and weak (COO(-)) cation exchange groups were thereafter introduced onto the GMA-grafted monoliths by reactions with sodium hydrogen sulfite and iminodiacetic acid, respectively. Grafting was confirmed by XPS, gravimetric measurement, and chromatographic behavior of the modified materials toward model proteins. In incubation experiments the proteins were recovered quantitatively with no obvious signs of unfolding after contact with the stationary phase for >2 h. Chromatographic assessments on the functionalized columns as well as problems associated with flow-through modification by ATRP are discussed.

Preparation and characterization of sizable macroporous epoxy resin-based monolithic supports for flow-through systems.

This paper presents further results from our efforts to prepare sizable macroporous monolithic materials from epoxy resins and polyamines by emulsion polymerization. For their uses as supports in flow systems, the study aimed at developing materials possessing maximum fluid permeability, high mechanical stability, and a controlled porosity and surface area. Characterization of the materials has been carried out using different techniques, focusing on morphological and mechanical features, and on the surface chemistry. Morphology and porosity were studied with SEM, nitrogen adsorption/desorption, mercury intrusion porosimetry (MIP), and (2)H NMR cryoporosimetry. The chemical composition of the bulk structures and their surfaces was studied by means of bulk elemental analysis and X-ray photoelectron spectroscopy, and potentiometric titration was used to assess the relative amounts of amines and epoxy groups. Essentially, the morphological features were a high fluid permeability, but rather low specific surface area. Convective flow was facilitated by large, interconnected, and evenly spaced macropores which were formed by nonporous skeletons of the connected-rod type. Despite the interfacial nature of the polymerization, the bulk and the surface of the fully cured materials showed similar elemental compositions. All materials were found to have a high surface density of hydroxyl groups, which facilitates functionalization reactions.

Interaction of toluene with polar stationary phases under conditions typical of hydrophilic interaction chromatography probed by saturation transfer difference nuclear magnetic resonance spectroscopy.

Toluene has been used as void volume (zero retention) marker since the inception of hydrophilic interaction chromatography (HILIC), based on the assumption that its hydrophobicity should prevent it from interacting with stationary phases envisioned to be covered by relatively thick layers of water. Recent work has shown that the void volumes of partly water-swollen HILIC phases are not identical to the volumes probed by toluene, yet the compound is still ubiquitously used as void volume marker. As part of our investigations of the retention mechanisms in HILIC, we probed the extent to which toluene is capable of penetrating into the water-enriched layer and to interact with the functional groups of three commercially available hydrophilic and polar stationary phases with different charge properties and water-retaining abilities, using saturation transfer difference 1H nuclear magnetic resonance (STD-NMR) spectroscopy at high resolution magic angle spinning (HR-MAS) conditions. The test solutions were 1000 ppm of toluene in deuterated acetonitrile and water mixtures, with and without addition of ammonium acetate, in order to mimic a set of conditions typically encountered in HILIC separations. Interactions between toluene and the functional groups on the stationary phases were probed by equilibrating the phases with these eluent mimics and measuring the transfer of magnetization from stationary phase protons to the protons of toluene. Our results show that toluene is indeed capable of traversing the water-enriched layers of all the three tested phases and of interacting with protons that are tightly associated with the stationary phases.

Preparation of a sorbitol methacrylate grafted silica as stationary phase for hydrophilic interaction chromatography.

A new highly hydrophilic stationary phase based on graft polymerization of sorbitol methacrylate from the surface of Kromasil silica particles is described. Polymerization was initiated by thermal cleavage of tert-butyl hydroperoxide covalently attached to the silica particle surface. Due to the highly amphiphilic properties of the monomer, an extensive search was needed to find solvent conditions that enabled surface-initiated polymerization. This was finally solved by using a mixture of solvents that only partially dissolved the monomer. The graft polymerization was confirmed by Fourier transform infrared spectroscopy and elemental analysis. The resulting stationary phase was evaluated by HPLC and exhibited a selectivity markedly different from that of commercially available columns and of neat silica.

Alternative organic solvents for HILIC separation of cisplatin species with on-line ICP-MS detection.

Several low volatile organic solvents were evaluated as organic modifiers in eluents for HILIC separations of cisplatin species to optimize the on-line coupling of HILIC to inductively coupled plasma MS (ICP-MS). The aim was to identify a solvent giving low solvent vapor loading of the ICP, to maximize analyte sensitivity and minimize carbon depositions on instrumental parts, while retaining chromatographic performance. The best overall performance of the HILIC-ICP-MS system for the analysis of cisplatin was achieved using 1,4-dioxane as eluent, yielding high retention and an HILIC type retention mechanism, at the expense of a 50% drop in column efficiency due to the higher viscosity of 1,4-dioxane compared to the more commonly used HILIC solvent ACN. Using 1,4-dioxane as solvent in HILIC provides the best compromise between carbon deposition and separation efficiency among a series of high-boiling water-miscible solvents tested.