Spectroscopy and quantum chemical modeling reveal a predominant contribution of excitonic interactions to the bathochromic shift in alpha-crustacyanin, the blue carotenoprotein in the carapace of the lobster Homarus gammarus

To resolve the molecular basis of the coloration mechanism of alpha-crustacyanin, we used (13)C-labeled astaxanthins as chromophores for solid-state (13)C NMR and resonance Raman spectroscopy of [6,6',7,7']-(13)C(4) alpha-crustacyanin and [8,8',9,9',10,10',11,11',20,20']-(13)C(10) alpha-crustacyanin. We complement the experimental data with time-dependent density functional theory calculations on several models based on the structural information available for beta-crustacyanin. The data rule out major changes and strong polarization effects in the ground-state electron density of astaxanthin upon binding to the protein. Conformational changes in the chromophore and hydrogen-bond interactions between the astaxanthin and the protein can account only for about one-third of the total bathochromic shift in alpha-crustacyanin. The exciton coupling due to the proximity of two astaxanthin chromophores is found to be large, suggesting that aggregation effects in the protein represent the primary source of the color change.     

Lanthanide luminescence quenching as a detection method in ion chromatography. Chromate in surface and drinking water

Dynamic quenching of Eu(III) and Tb(III) luminescence by inorganic anions as a detection method in ion chromatography was investigated. To obtain a high luminescence intensity, lanthanide(III) complexes are formed with ligands which make indirect excitation of the ions possible. Only a few anions (e.g., nitrite, chromate) induce efficient dynamic luminescence quenching. Chromate is an efficient quencher of Tb-acac luminescence. Samples of tap water and surface water, spiked with chromate, were injected into a high-performance liquid chromatographic system with post-column addition of the luminescent complex. In this way, a detection limit of 1.1 . 10(-7) M (13 ppb) of chromate could be obtained.     

Forward-scattering degenerate four-wave mixing as a potential laser-based absorption detection method in liquid separation systems: Coupling to conventional-size liquid chromatography

An explorative study on the compatibility of liquid separation systems, such as (micro) liquid chromatography (LC) and capillary electrophoresis (CE), and forward-scattering degenerate four-wave mixing (F-D4WM) as a detection method is presented. F-D4WM is a laser-based technique showing some analogy with holographic spectroscopy: a signal on a theoretical dark background is observed as a result of light absorption by an analyte. Parameters considered are solvent composition focussing on acetonitrile, methanol and water; mobile phases in LC and CE), detector cell construction, and influences of laser beam powers. A specially designed detector cell has been developed to meet the Brewster condition, both at the air-quartz and the quartz-liquid boundaries. For practical reasons, the tested cell has an optical pathlength of 1 mm; reduction to 100 μm is required to apply the cell in microseparations. The F-D4WM technique has been involved for detection in a conventional-size, reversed-phase LC separation of 1- and 2-aminoanthraquinones. The detection limit obtained (for the 1 mm cell) is 2 × 10⁻⁵ absorbance units. The experiments indicate that further reduction of background deserves explicit attention.     

Shpol''skii fluorimetric determination of polycyclic aromatic hydrocarbons in complex environmental samples

Summary High-resolution fluorimetry in low-temperature n-alkane Shpol'skii matrices is a powerful technique for the analysis of rigid, non-polar compounds like polycyclic aromatic hydrocarbons. Because of the method's sensitivity and selectivity, sample clean-up, preconcentration and even chromatographic separation can often be left out. The Shpol'skii analysis of pyrene in crude extracts from marine sediments and from bird meat is demonstrated. Special attention is focussed on the extra possibilities acquired when a laser is used as excitation source (Laser Excited Shpol'skii Spectroscopy, LESS).     

Raman imaging of PLGA microsphere degradation inside macrophages

Understanding the degradation behavior of polymeric microspheres is crucial for the successful application of such devices in controlled drug delivery. The degradation mechanism of poly(lactic-co-glycolic acid) (PLGA) microspheres inside phagocytic cells is not known, but different models for degradation in aqueous solution have been proposed. We have used confocal Raman spectroscopy and imaging to study the intracellular degradation of PLGA microspheres inside individual macrophages. Our results show that ingested microspheres degrade in a heterogeneous manner, with a more rapid degradation in the center. Comparison of Raman spectra from degrading beads with those of uningested beads reveals that ester hydrolysis occurs throughout the phagocytosed microspheres, with a selective loss of glycolic acid units. Furthermore, we show that PLGA degradation is a cell-mediated process, possibly caused by the low pH of the phagosome and/or the presence of hydrolytic enzymes. In conclusion, we have demonstrated that the chemical composition of degrading polymers inside cells can be probed by Raman spectral imaging. This technique will expand the capabilities of investigating biomaterial degradation in vivo.     

Thermostating in capillary electrophoresis

Summary The use of high voltages across a electrophoresis capillary will increase the temperature of the buffer due to Joule heating. As a result temperature control in CE is rather important since variations in the buffer temperature will result in changes in the pH of the buffer, peak shape, migration time, reproducibility, efficiency, 3-D structure of macromolecular analytes, etc. Six different thermostating systems have been evaluated: (i) natural convection, (ii) fan, (iii) home-made and (iv and v) two commercially available high-speed air and a (vi) liquid thermostated device. In all cases the temperature of the buffer in the capillary is calculated according to the temperature-conductivity relationship. For this purpose two parameters are introduced describing temperature control: the temperature onset (δT) and the temperature rise factor (α). From these results, it can be concluded that high speed air thermostating can be as efficient as liquid thermostating.     

Laser-Induced fluorescence detection in conventional-size liquid chromatography with a frequency-doubled argon-ion laser

Laser-induced fluorescence (LIF) detection in conventional-size column liquid chromatography is achieved at 257 nm with a frequency-doubled argon-ion laser. Short-wavelength excitation offers two important advantages: firstly, a wide variety of analytes can be excited, and secondly, the Raman scatter of the eluent does not interfere with the fluorescence of the analytes. A standard mixture of polynuclear aromatic hydrocarbons was studied, both with LIF detection and with a commercially available sensitive conventional fluorescence detector. The improvement in the detection limits ranges from about a factory of 4 to 30; the LIF detection limits are typically at the 50 ng l^?1 level, which corresponds to an injected amount of 0.5 pg.     

Coupling of narrow-bore liquid chromatography to thin-layer chromatography : Part I. Interfacing

The coupling of liquid chromatography (l.c.) on narrow-bore columns to thin-layer chromatography (t.l.c.) is described. The effluent from a l.c. column can be deposited on a t.l.c. plate after a normal-phase or reversed-phase separation without serious loss of chromatographic information. Both silica and alkyl-modified silica plates can be used for storage. The interface is a fused silica capillary which connects the column outlet to the spray jet assembly of a Linomat applicator for t.l.c. The stored chromatogram can serve as starting point for a new separation, but also allows the use of detection principles which are normally not compatible with l.c. The chromatography of some polynuclear aromatic hydrocarbons is used to illustrate the possibilities of the combinations.     

A Titration Microcalorimetric Study of the Effects of Halide Counterions on Vesicle-Forming Aggregation in Aqueous Solution of Branched-Chain Alkylpyridinium Surfactants

Titration microcalorimetry is used to study the influences of iodide, bromide, and chloride counterions on the aggregation of vesicle-forming 1-methyl-4-(2-pentylheptyl)pyridinium halide surfactants. Formation of vesicles by these surfactants was characterised using transmission electron microscopy. When the counterion is changed at 303 K through the series iodide, bromide, to chloride, the critical vesicular concentration (cvc) increases and the enthalpy of vesicle formation changes from exo- to endothermic. With increase in temperature to 333 K, vesicle formation becomes strongly exothermic. Increasing the temperature leads to a decrease in enthalpy and entropy of vesicle formation for all three surfactants. However the standard Gibbs energy for vesicle formation is, perhaps surprisingly, largely unaffected by an increase in temperature, as a consequence of a compensating change in both standard entropy and standard enthalpy of vesicle formation. Interestingly, standard isobaric heat capacities of vesicle formation are negative, large in magnitude but not strikingly dependent on the counterion. We conclude that the driving force for vesicle formation can be understood in terms of overlap of the thermally labile hydrophobic hydration shells of the alkyl chains. Copyright 2000 Academic Press.