Characterization of Coupled Ground State and Excited State Equilibria by Fluorescence Spectral Deconvolution

Fluorescence probes with multiparametric response based on the relative variation in the intensities of several emission bands are of great general utility. An accurate interpretation of the system requires the determination of the number, positions and intensities of the spectral components. We have developed a new algorithm for spectral deconvolution that is applicable to fluorescence probes exhibiting a two-state ground-state equilibrium and a two-state excited-state reaction. Three distinct fluorescence emission bands are resolved, with a distribution of intensities that is excitation-wavelength-dependent. The deconvolution of the spectrum into individual components is based on their representation as asymmetric Siano-Metzler log-normal functions. The application of the algorithm to the solvation response of a 3-hydroxychromone (3HC) derivative that exhibits an H-bonding-dependent excited-state intramolecular proton transfer (ESIPT) reaction allowed the separation of the spectral signatures characteristic of polarity and hydrogen bonding. This example demonstrates the ability of the method to characterize two potentially uncorrelated parameters characterizing dye environment and interactions.     

Validation of an analytical method to determine trace metal impurities in fluoride compounds by flame atomic absorption spectroscopy

 The development of an analytical method for the determination of some heavy metals (Fe, Cu, Co, Zn and Ni) in fluoride compounds [Cu(BF₄)₂, Sn(BF₄)₂, Pb(BF₄)₂ and HBF₄] by flame atomic absorption spectroscopy is described. This method is to be used as a routine analytical method in an industrial quality control laboratory. To this end the "performance characteristics" of an instrumental analytical method such as matrix effects, sensitivity, linearity, detection and quantitation limits, precision and accuracy were evaluated for every system under study. The results of these investigations showed that non-spectral interferences (due to the presence of large concentrations of major metals such as Cu, Sn and Pb) were observed. Nevertheless it was possible to define a matrix concentration interval where matrix effects were not statistically significant, and therefore a direct calibration approach could be used as the calibration tool whenever the major metal concentration was not higher than 40×10^–3 kg l^–1. A guide to the developement of an analytical method for trace metal determination is provided. General tools for quality control have been used in order to show how an analytical method can be tested daily and evaluated in a convenient manner. Received: 29 January 1997 Accepted: 11 March 1997     

Single-walled carbon nanohorns immobilized on a microporous hollow polypropylene fiber as a sorbent for the extraction of volatile organic compounds from water samples

We have evaluated the behavior of single-walled carbon nanohorns as a sorbent for headspace and direct immersion (micro)solid phase extraction using volatile organic compounds (VOCs) as model analytes. The conical carbon nanohorns were first oxidized in order to increase their solubility in water and organic solvents. A microporous hollow polypropylene fiber served as a mechanical support that provides a high surface area for nanoparticle retention. The extraction unit was directly placed in the liquid sample or the headspace of an aqueous standard or a water sample to extract and preconcentrate the VOCs. The variables affecting extraction have been optimized. The VOCs were then identified and quantified by GC/MS. We conclude that direct immersion of the fiber is the most adequate method for the extraction of VOCs from both liquid samples and headspace. Detection limits range from 3.5 to 4.3 ng L^?1 (excepted for toluene with 25 ng L^?1), and the precision (expressed as relative standard deviation) is between 3.9 and 9.6 %. The method was applied to the determination of toluene, ethylbenzene, various xylene isomers and styrene in bottled, river and tap waters, and the respective average recoveries of spiked samples are 95.6, 98.2 and 86.0 %.

Potential of supramolecular solvents for the extraction of contaminants in liquid foods

Amphiphile-based supramolecular solvents (ASSs), which are water immiscible liquids consisting of supramolecular aggregates in the nano- and micro-scale regimes dispersed in a continuous phase, were assessed for the extraction of trace contaminants in liquid foods. The ASS selected was made up of reversed micelles of decanoic dispersed in tetrahydrofuran (THF)-water and the contaminants used as a model were bisphenol A (BPA), ochratoxin A (OTA) and benzo(a)pyrene (BaPy). The influence of matrix components on the extractant solvent production, extraction recoveries and actual concentration factors was investigated by using commercial foods such as wine and wine-based products, beer, soft drinks and tea and coffee brews, and/or aqueous synthetic solutions containing specific food matrix components. The method involved the addition of decanoic acid (80mg) and THF (0.8-1.7mL) to the food sample (15mL), stirring of the mixture for 5min, centrifugation for 10min and analysis of 10-20microL of the extract by liquid chromatography coupled to fluorimetry for OTA and BaPy or to mass spectrometry for BPA. No clean-up of the crude extracts was required for any of the samples analysed. The quantification limits for the contaminants (14-31ngL(-1), 0.37-0.39ngL(-1) and 562-602ngL(-1) for OTA, BaPy and BPA, respectively) were far below their respective European legislative threshold limits. Recoveries for food samples were in the ranges 79-93%, 90-96% and 78-82% for OTA, BaPy and BPA, respectively, with relative standard deviations ranging from 1 to 7%, and actual concentrations factors between 65 and 141. The methods developed were applied to the determination of the target compounds in a variety of commercial foods. OTA was found in vinegar, must and beer samples, the concentrations ranging from 92 to 177ngL(-1), BaPy was quantified in samples of tea and coffee at concentrations between 1.5 and 16.6ngL(-1) whereas BPA was detected in two canned soft drinks and quantified in one of them (tea beverage) at a level of 2.3microgL(-1).     

Computational and experimental structure–reactivity relationships: evidence for a side reaction in Alpine-Borane reductions of d-benzaldehydes

Extraordinary stereoselectivity, approaching 100%, has been reported in the reductions of d-benzaldehydes by B-isopinocampheyl-9-borabicyclo[3.3.1]nonane (Alpine-Borane). This is likely because of the extreme size disparity of groups on either side of the carbonyl. Here, we present a structure–reactivity study whereby the reductions of variably substituted d-benzaldehydes are explored using highly sensitive measures for enantiomeric excess and relative reactivity. These results are compared to the relative rates predicted from density functional calculations. The results indicate that 2,6-disubstitution adversely affects the stereoselectivity by means of a non-selective reduction via the dehydroboration product of Alpine-Borane, 9-borabicyclo[3.3.1]nonane.     

Characteristics and safety of nano-sized cellulose fibrils

A finely ground fibrillated cellulose was fractionated into separate size fractions. The characteristics of the smallest size fractions were studied, and the toxicity to humans was tested as part of a safety assessment. Morphological studies performed with state-of-the-art methods, such as scanning electron microscopy and atomic force microscopy, showed that the fraction obtained consisted of long thin fibrils but also larger fibril agglomerates, and spherical particles were present. The finest fraction did not show any sub-lethal effects as assessed by RNA inhibition test in vitro, nor were there any indications of genotoxicity as tested by the Ames test in vitro. Systemic effects tested in vivo with the nematode were also absent. No cytotoxic effects were seen in the highest tolerated dose test in vitro, but some indication of cytotoxicity was observed in the total protein content test in vitro at the highest sample concentration. The significance of this toxicity test result should be addressed in relation to the other toxicity tests, in which no toxicity was observed, with special emphasis on the in vivo test. Given this, the overall toxicity analyses support the conclusion that nano-scale cellulose fibrils can be considered to be safe towards humans. However, the reason for the positive cytotoxicity test result and, in addition, the effect of the biocide used in sample preservation on the toxicity tests need to be clarified before generalizing these results and declaring nanocellulose to be unambiguously safe.     

Tetrahydrofuran–water extraction,in-line clean-up and selective liquid chromatography/tandem mass spectrometry for the quantitation of perfluorinated compounds in food at the low picogram per gram level

A new solvent extraction system was developed for extraction of PFCs from food. The extraction is carried out with 75:25 (v/v) tetrahydrofuran:water, a solvent mixture that provides an appropriate balance of hydrogen bonding, dispersion and dipole–dipole interactions to efficiently extract PFCs with chains containing 4–14 carbon atoms from foods. This mixture provided recoveries above 85% from foods including vegetables, fruits, fish, meat and bread; and above 75% from cheese. Clean-up with a weak anion exchange resin and Envi-carb SPE, which were coupled in line for simplicity, was found to minimize matrix effects (viz. enhancement or suppression of electrospray ionization). The target analytes (PFCs) were resolved on a perfluorooctyl phase column that proved effective in separating mass interferences for perfluorooctane sulfonate (PFOS) in fish and meat samples. The mass spectrometer was operated in the negative electrospray ionization mode and used to record two transitions per analyte and one per mass-labeled method internal standard. The target PFCs were quantified from solvent based calibration curves. The limits of detection (LODs) were as low as 1–5 pg analyte g⁻¹ food; by exception, those for C₄ and C₅ PFCs were somewhat higher (25–30 pg g⁻¹) owing to their less favourable mass response. To the best of our knowledge these are among the best LODs for PFCs in foods reported to date. The analysis of a variety of foods revealed contamination with PFCs at levels from 4.5 to 75 pg g⁻¹ in 25% of samples (fish and packaged spinach). C₁₀–C₁₄ PFCs were found in fish, which testifies to the need to control long-chain PFCs in this type of food. The proposed method is a useful tool for the development of a large-scale database for the presence of PFCs in foods.     

Conformational dependence of pyranoid rings 1,2-cis-fused to dioxolane rings on the configuration of the dioxolane rings in acetylated derivati

X-Ray and ¹H N.M.R. studies on pyranoid rings 1,2-$\text{cis}$-fused to dioxolane rings in acetylated $\text{D}$-gluco- and $\text{D}$--galactopyranose derivatives demonstrate that the configuration of the dioxolane ring influences the conformation of the pyranoid ring in the $\text{D}$-gluco but not in the $\text{D}$-galactopyranose series. The crystal structure of 3,4,6-tri-$\text{O}$-acetyl-1,2-$\text{O}$-(R)--(l-cyano-ethylidene)-α-$\text{D}$-glucopyranose ($\text{1}$) and 3,4,6-tri-$\text{O}$-acetyl-1,2-$\text{O}$-($\text{R}$)-(1-cyano-ethylidene)-α-$\text{D}$-galactopyranose ($\text{2}$)have been determined by X-ray analysis. Lattice parameters for $\text{1}$ are a=20.6021 (11), b=8.0438 (2), c=5.5541 (1) Å and β= 95.588 (3)° for a cell with P21 symmetry. These parameters for $\text{2}$ are a=20.3361 (7), b=10.0907 (2), c=18.9115 (5) Å, β =112.399 (2)°, C2, with two crystallographycally independent molecules. The conformation of the pyranoid ring in both compounds can be described as flattened ⁴C₁ and that of the dioxolane ring as distorted E₁. The importance of the torsion angles for describing problems of configuration is remarked and the use of relative configurational angles is stressed. The ¹H N.M.R. spectra of $\text{1}$ and $\text{2}$ and 3,4,6-tri-$\text{O}$-acetyl-1,2-O-(S)- and (R)-ethylidene-α-$\text{D}$-glucopyranose ($\text{5}$ and $\text{7}$), 3,4,6-tri-O-acetyl--1,2-O-(S)- and ($\text{R}$)-ethylidene-α-$\text{D}$-galactopyranose ($\text{6}$ and $\text{8}$), and 3,4,6-tri-$\text{O}$-acetyl-1,2-$\text{O}$-($\text{S}$)-and ($\text{R}$)-benzylidene-α-$\text{D}$-glucopyranose ($\text{9}$ and $\text{10}$) have been analyzed by using iterative computer methods and N.O.E. measurements. The results indicate that the major solution conformation of the pyranoid ring of the derivatives in the $\text{D}$-gluco series $\text{1}$, $\text{5}$ and $\text{9}$ may be described as flattened ⁴C₁ and that of $\text{7}$ and $\text{10}$ as ²$\text{S}$₅. The major solution conformation of the pyranoid ring in all compounds in the $\text{D}$-galacto series ($\text{2}$,$\text{4}$,$\text{6}$,$\text{8}$) may be described as flattened ⁴$\text{C}$₁.     

Small molecule fluorescent probes for the detection of amyloid self-assembly in vitro and in vivo

The misfolding and aggregation of amyloidogenic polypeptides are characteristics of many neurodegenerative syndromes including Alzheimer's and Parkinson's disease. There is a major interest in the availability of amyloid-specific probes that exhibit fluorescence properties, for its use as reporters of protein aggregation in spectroscopy and microscopy methodologies. In this review, we intend to provide an overview of novel fluorescence-based probes and procedures applied for addressing fundamental aspects of amyloid self-assembly in vitro and in vivo. We highlight the utilization in vitro of several small-molecule fluorescent probes as extrinsic and site-specific reporters of amyloid formation, including single-molecule determinations. Detection of amyloid self-assembly employing compounds such as JC-1, DCVJ, ANS derivatives and luminescent conjugated polymers, as well as site-specific probes such as pyrene and ESIPT is discussed. We further review novel fluorescent probes developed for the non-invasive optical imaging of protein aggregates in vivo, including BTA-1, Methoxy-X04, NIAD-4 and CRANAD-2. Availability of increasingly versatile amyloid-specific fluorescent probes is having a very positive impact in the drug discovery and diagnostics fields.