Multiple Step Gradient Analysis in Stationary Phase Optimised Selectivity LC for the Analysis of Complex Mixtures

Stationary phase optimised selectivity liquid chromatography (SOSLC) is an approach to tune a given LC separation by combining different stationary phases in a multi- segment column set-up. The presently available SOSLC optimisation procedure and algorithm are, however, only applicable to isocratic conditions. This is a severe limitation for the analysis of mixtures composed of components covering a broad hydrophobicity range. A strategy is described to circumvent this limitation. The components of a mixture are divided into different groups according to hydrophobicity as elucidated by a gradient analysis on a C₁₈ reversed-phase column. Each group separation is then individually optimised with a specific isocratic mobile phase composition using the original SOSLC strategy. The mobile phase composition thereby only differs in the percentage of organic modifier between the various groups. Finally, a combination of stationary phases that guarantees sufficient selectivity for all the groups is selected and the separation is performed by a multiple step gradient, whereby each level consists of the mobile phase composition applied for the SOSLC optimisation of the individual groups. The multi step gradient approach is demonstrated through the analysis of a mixture of 27 steroids covering a wide range of hydrophobicity.     

Synthesis of 7-(E)-alkenyl-4-amino-3-quinolinecarbonitriles via Pd-mediated Heck, Stille, and Suzuki reactions

A regio- and stereoselective synthesis of 7-(E)-alkenyl-4-amino-3-quinolinecarbonitriles via Pd-mediated coupling reactions was developed. The comparison and optimization of stereoselectivity of the Heck, Stille, and Suzuki reactions of 7-bromo or 7-triflate-3-quinolinecarbonitrile are described. Compound 7 and 10 were potent inhibitors of Src kinase and Raf/Mek activity, respectively.     

Subnanomolar detection limit application of ion-selective electrodes with three-dimensionally ordered macroporous (3DOM) carbon solid contacts

Solid-contact ion-selective electrodes (SC-ISEs) can exhibit very low detection limits and, in contrast to conventional ISEs, do not require an optimization of the inner filling solution. This work shows that subnanomolar detection limits can also be achieved with SC-ISEs with three-dimensionally ordered macroporous (3DOM) carbon contacts, which have been shown recently to exhibit excellent long-term stabilities and good resistance to the interferences from oxygen and light. The detection limit of 3DOM carbon-contacted electrodes with plasticized poly-(vinyl chloride) as membrane matrix can be improved with a high polymer content of the sensing membrane, a large ratio of ionophore and ionic sites, and conditioning with a low concentration of analyte ions. This permits detection limits as low as 1.6 × 10⁻⁷ M for K⁺ and 4.0 × 10⁻¹¹ M for Ag⁺.     

Matisse to Picasso: a compositional study of modern bronze sculptures

Inductively coupled plasma-optical emission spectroscopy (ICP-OES) was used to determine the bulk metal elemental composition of 62 modern bronze sculptures cast in Paris in the first half of the twentieth century from the collections of The Art Institute of Chicago and the Philadelphia Museum of Art. As a result, a comprehensive survey of the alloy composition of the sculptures of many prominent European artists of the early twentieth century is presented here for the first time. The sculptures in this study consist of predominantly copper with two main alloying elements (zinc and tin). By plotting the concentrations of these two elements (zinc and tin) against each other for all the sculptures studied, three clusters of data become apparent: (A) high-zinc brass; (B) low-zinc brass; (C) tin bronze. These clusters correlate to specific foundries, which used specific casting methods (sand or lost wax) that were influenced by individual preferences and technical skills of the foundry masters. For instance, the high-zinc brass alloys (with the highest levels of tin and zinc and the lowest melting temperature) correspond to most of the Picasso sculptures, correlate with the Valsuani foundry, and are associated with the most recent sculptures (post-WWII) and with the lost-wax casting method. By expanding the ICP-OES database of objects studied, these material correlations may become useful for identifying, dating, or possibly even authenticating other bronzes that do not bear foundry marks. Figure   Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Fluorescence resonance energy transfer (FRET) for DNA biosensors: FRET pairs and Förster distances for various dye-DNA conjugates

Fluorescence resonance energy transfer (FRET) between the extrinsic dye labels Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethyl Rhodamine (TAMRA), Iowa Black Fluorescence Quencher (IabFQ), and Iowa Black RQ (IabRQ) has been studied. The F?rster distances for these FRET-pairs in single- and double-stranded DNA conjugates have been determined. In particular, it should be noted that the quantum yield of the donors Cy3 and TAMRA varies between single- and double-stranded DNA. While this alters the F?rster distance for a donor-acceptor pair, this also allows for detection of thermal denaturation events with a single non-intercalating fluorophore. The utility of FRET in the development of nucleic acid biosensor technology is illustrated by using TAMRA and IabRQ as a FRET pair in selectivity experiments. The differential quenching of TAMRA fluorescence by IabRQ in solution has been used to discriminate between 0 and 3 base pair mismatches at 60 degrees C for a 19 base sequence. At room temperature, the quenching of TAMRA fluorescence was not an effective indicator of the degree of base pair mismatch. There appears to be a threshold of duplex stability at room temperature which occurs beyond two base pair mismatches and reverses the observed trend in TAMRA fluorescence prior to that degree of mismatch. When this experimental system is transferred to a glass surface through covalent coupling and organosilane chemistry, the observed trend in TAMRA fluorescence at room temperature is similar to that obtained in bulk solution, but without a threshold of duplex stability. In addition to quenching of fluorescence by FRET, it is believed that several other quenching mechanisms are occurring at the surface.     

Stability of pesticides on solid-phase extraction disks after incubation at various temperatures and for various time intervals: interlaboratory study

An interlaboratory study was conducted at 8 locations to assess the stability of pesticides on solid-phase extraction (SPE) disks after incubation at various temperatures and for various time intervals. Deionized water fortified with selected pesticides was extracted by using 2 types of SPE filtration disks (Empore C18 and Speedisk C18XF), and after extraction, the disks were incubated at 3 temperatures (25, 40, and 55 degrees C) and for 2 time intervals (4 and 14 days). Deionized water was fortified with atrazine, carbofuran, and chlorpyrifos by all participating laboratories. In addition, some of the laboratories included 2 of the following pesticides: metolachlor, metribuzin, simazine, chlorothalonil, and malathion. Concurrently, fortified water samples were extracted with the incubated samples by using each disk type at 4 and 14 days. Pesticides had equivalent or greater stability on > or = 1 of the C18 disk types, compared with storage in water. The lowest recoveries of carbofuran (6%) and chlorpyrifos (7%) were obtained at 55 degrees C after storage for 14 days in incubated water. At 55 degrees C after 14 days, the lowest recovery for atrazine was 65% obtained by using Empore disks. Pesticide-specific losses occurred on the C18 disks in this study, underlining the importance of temperature and time interval when water is extracted at remote field locations and the SPE disks containing the extracted pesticides are transported or shipped to a laboratory for elution and analysis.     

Sequence requirements and an optimization strategy for short antimicrobial peptides

Short antimicrobial host-defense peptides represent a possible alternative as lead structures to fight antibiotic resistant bacterial infections. Bac2A is a 12-mer linear variant of the naturally occurring bovine host defense peptide, bactenecin, and demonstrates moderate, broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria as well as against the yeast Candida albicans. With the assistance of a method involving peptide synthesis on a cellulose support, the primary sequence requirements for antimicrobial activity against the human pathogen Pseudomonas aeruginosa of 277 Bac2A variants were investigated by using a luciferase-based assay. Sequence scrambling of Bac2A led to activities ranging from superior or equivalent to Bac2A to inactive, indicating that good activity was not solely dependent on the composition of amino acids or the overall charge or hydrophobicity, but rather required particular linear sequence patterns. A QSAR computational analysis was applied to analyze the data resulting in a model that supported this sequence pattern hypothesis. The activity of selected peptides was confirmed by conventional minimal inhibitory concentration (MIC) analyses with a panel of human pathogen bacteria and fungi. Circular-dichroism (CD) spectroscopy with selected peptides in liposomes and membrane depolarization assays were consistent with a relationship between structure and activity. An additional optimization process was performed involving systematic amino acid substitutions of one of the optimal scrambled peptide variants, resulting in superior active peptide variants. This process provides a cost and time effective enrichment of new candidates for drug development, increasing the chances of finding pharmacologically relevant peptides.     

Redox magnetohydrodynamic enhancement of stripping voltammetry: toward portable analysis using disposable electrodes, permanent magnets, and small volumes

The use of redox magnetohydrodynamics (MHD) to enhance the anodic stripping voltammetry (ASV) response of heavy metals has been investigated, with respect to achieving portability: disposable electrodes consisting of screen-printed carbon (SPC) on a low temperature co-fired ceramic (LTCC) substrate, small volumes, and permanent magnets. The analytes tested (Cd(2+), Cu(2+), and Pb(2+)) were codeposited on SPC with Hg(2+) to form a Hg thin film electrode. High concentrations of Fe(3+) were used to produce a high cathodic current which generates a significant Lorentz force in the presence of a magnetic field. This Lorentz force induces solution convection during the deposition step, enhancing the mass transport of analytes to the electrode and increasing their preconcentrated quantity in the mercury thin film. Therefore, larger ASV peaks and improved sensitivities are obtained, compared to analyses performed without a magnet. The effects on ASV signal of varying Hg(2+) concentration (0.10 and 1.0 mM), deposition time (10-600 s), and electrode surface roughness were investigated. In addition, analyses were performed using a real lake water matrix. By using the disposable LTCC-SPC working electrodes in small volumes (150 microL) and with small permanent magnets (0.78 T), peak areas were increased by 75% when compared to the signal obtained in the absence of a magnetic field. A limit of detection of 25 nM for Cd(2+) was observed with only a 1 min preconcentration time.     

A 1H/19F minicoil NMR probe for solid-state NMR: application to 5-fluoroindoles

We show that it is feasible to use a minicoil for solid-state 19F 1H NMR experiments that has short pulse widths, good RF homogeneity, and excellent signal-to-noise for small samples while using low power amplifiers typical to liquid-state NMR. The closely spaced resonant frequencies of 1H and 19F and the ubiquitous use of fluorine in modern plastics and electronic components present two major challenges in the design of a high-sensitivity, high-field 1H/19F probe. Through the selection of specific components, circuit design, and pulse sequence, we were able to build a probe that has low 19F background and excellent separation of 1H and 19F signals. We determine the principle components of the chemical shift anisotropy tensor of 5-fluoroindole-3-acetic acid (5FIAA) and 5-fluorotryptophan. We also solve the crystal structure of 5FIAA, determine the orientation dependence of the chemical shift of a single crystal of 5FIAA, and predict the 19F chemical shift based on the orientation of the fluorine in the crystal. The results show that this 1H/19F probe is suitable for solid-state NMR experiments with low amounts of biological molecules that have been labeled with 19F.     

Direct electrospray tandem mass spectrometry of the unstable hydroperoxy bishemiacetal product derived from cholesterol ozonolysis

Cholesterol is the most abundant neutral lipid in the epithelial lining fluid of the lower airways of the lung also known as pulmonary surfactant and a potential target for reaction with ambient ozone when inspired into the human lung. The isolated double bond of cholesterol has been shown to be susceptible to attack by ozone, but the major reaction product from cholesterol ozonolysis had been remarkably difficult to structurally characterize. Recently, NMR and X-ray crystallography have been used to suggest the formation of a hydroperoxy, hydroxy hemiacetal product, using various derivatives and models of cholesterol to stabilize this chemically reactive product. Electrospray ionization mass spectrometry was used to study the somewhat unstable ozonolysis product of cholesterol which was found to display unique ionization and fragmentation properties when collisionally activated. The electron-deficient carbon atoms of this highly oxygenated product permitted covalent attachment of an acetate anion during negative ion electrospray ionization, leading to the formation of abundant adduct ions at m/z 511. Surprisingly, positive ions were not readily formed. Collision induced dissociation of the adduct anion yielded a major ion at m/z 477, corresponding to the loss of hydrogen peroxide. The most abundant fragment ion following collisional activation was observed at m/z 93, resulting from a complex rearrangement subsequent to the attack of the hydroperoxide anion on the carbon center of the acetate adduct. Based on the interpretation of the tandem mass spectral data, the major cholesterol ozonization product was characterized as a hydroperoxy, hydroxy hemiacetal derivative, which was consistent with the NMR and X-ray crystallographic studies which were carried out on the more stable methyl ether derivative.