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The use of a state-of-the-art commercial solvent-elimination interface for liquid chromatography-infrared spectroscopy is discussed from the perspective of quantitative analysis. The effect of eluent flow-rate is investigated with respect to the homogeneity of the deposit and the trace width along the deposition trace. Low flow-rates (50 μl/min or less) turn out to be favorable for obtaining a good trace and a high sensitivity. The trace width decreased from 2.20 to 1.13 mm when the flow-rate was reduced from 500 to 25 μl/min. Preservation of chromatographic detail during deposition was evaluated at different substrate moving speeds. The additional (extra-column) band broadening that is inherent to nebulizer-deposition interfaces, causes a loss in resolution in size-exclusion chromatography (SEC)-Fourier transform infrared (FTIR) spectroscopy in comparison with SEC-UV. The repeatability of the deposition is evaluated by SEC-FTIR analyses of polystyrene standards (peak molar-masses: 1250-2 950 000 g/mol) and the RSD is found to range from 3.2 to 5.8% in response and from 0.21 to 0.47% in retention time. © 2002 Elsevier Science B.V. All rights reserved. Chemicals/CAS: Indicators and Reagents; Polystyrenes; Solvents

A liquid chromatographic method with evaporative mass detection (EMD) is described for the determination of paraffins in food contact materials that do not contain polyolefin oligomers, or paraffins migrating from these materials into fatty food simulants or certain simple foods. A normal-phase column operating at maximum column efficiency separates nonparaffinic and paraffinic materials without resolving the latter into individual components, and EMD is used to quantitate the paraffins. An on-line qualitative method that uses liquid chromatography/gas chromatography with flame ionization detection discriminates between paraffin waxes and oils in food contact materials, food simulants, and certain simple foods; a Fourier transform infrared spectrophotometric qualitative method also discriminates between waxes and oils, but is usually restricted to food contact materials that do not contain polyolefins and to migration experiments with organic solvents as fatty food simulants (with some other fatty food simulants, paraffin type must then be identified in the food contact material). Chemicals/CAS: Paraffin, 8002-74-2Chemicals/CAS: Paraffin, 8002-74-2

Heterologous conjugates of wheat arabinoxylan and β-casein were prepared via enzymatic cross-linking, using sequential addition of the arabinoxylan to a mixture of β-casein, peroxidase, and hydrogen peroxide. The maximal formation of adducts between the β-casein and the feruloylated arabinoxylan was reached at a protein-to-arabinoxylan ratio of 10:1, in combination with a molar ratio hydrogen peroxide to substrate of 2:1 and a molar protein-to-enzyme ratio between 102 and 104. The protein-arabinoxylan adducts were separated from the arabinoxylan homopolymers by size exclusion and anion exchange chromatography. The molar ratio protein:arabinoxylan in the purified conjugates varied between 0.1 and 5.6. This is the first report on the large-scale enzymatic preparation of heterologous protein-arabinoxylan conjugates.

Infrared-spectroscopic studies on the [NiFe]-hydrogenase of Chromatium vinosum-enriched in 15N or 13C, as well as chemical analyses, show that this enzyme contains three non-exchangeable, intrinsic, diatomic molecules as ligands to the active site, one carbon monoxide molecule and two cyanide groups. The results form an explanation for the three non-protein ligands to iron detected in the crystal structure of the Desulfovibrio gigas hydrogenase (Volbeda, A., Garcin, E., Piras, C., De Lacey, A. I., Fernandez, V. M., Hatchikian, E. C., Frey, M., and Fontecilla-Camps, J. C. (1996) J. Am. Chem. Soc. 118, 12989-12996) and for the low spin character of the lone ferrous iron ion observed with Mossbauer spectroscopy (Surerus, K. K., Chen, M., Van der Zwaan, W., Rusnak, F. M., Kolk, M., Duin, E. C., Albracht, S. P. J., and Munck, E. (1994) Biochemistry 33, 4980-4993). The results do not support the notion, based upon studies of Desulfovibrio vulgaris [NiFe]-hydrogenase (Higuchi, Y., Yagi, T., and Noritake, Y. (1997) Structure 5, 1671-1680), that SO is a ligand to the active site. The occurrence of both cyanide and carbon monoxide as intrinsic constituents of a prosthetic group is unprecedented in biology.

Two commercial liquid chromatography-Fourier-transform infrared spectroscopy interfaces (LC-FTIR), viz. a flow cell and a solvent-elimination interface have been assessed for use in size-exclusion chromatography (SEC) with respect to their chromatographic integrity (i.e. peak asymmetry, chromatographic resolution), quantitative and qualitative aspects. A polycarbonate/aliphatic polyester (PC/APE) blend and a polycarbonate-co-polydimethylsiloxane (PC-co-PDMS) copolymer were selected for the assessment. Both samples were successfully and selectively analyzed. The relatively large volume of the flow cell and the inherent deposition characteristics of the solvent-elimination interface led to a comparable decrease in the chromatographic resolution. The separation of oligomers was diminished in comparison with SEC-ultra-violet (UV). However, the peak asymmetry was not significantly affected by either interface. For both interfaces, a linear relationship was obtained for the FTIR response versus the injected concentration. The sensitivity was found to be higher for the solvent-elimination interface. For the current model compounds, the flow-cell interface detection limits are worse. However, the repeatability of flow-cell SEC-FTIR, evaluated by means of four SEC-FTIR analyses of polycarbonate, was considerably better than for solvent-elimination SEC-FTIR. This is probably due to the well-defined optical path length of the sample in the flow cell. By spectral subtraction, it was very well possible to obtain qualitative (functional group) information for compound identification also with flow-cell SEC-FTIR. © 2003 Elsevier B.V. All rights reserved.

Spectroscopy is a fast and rich analytical tool. On many occasions, spectra are acquired of two or more sets of samples that differ only slightly. These data sets then need to be compared and analyzed, but sometimes it is difficult to find the differences. We present a simple and effective method that detects and extracts new spectral features in a spectrum coming from one set with respect to spectra of another set on the basis of the fact that these new spectral features are essentially positive quantities. The proposed procedure (i) characterizes the spectra of the reference set by a component model and (ii) uses asymmetric least squares (ASLS) to find differences with respect to this component model. It should be stressed that the method only focuses on new features and does not trace relative changes of spectral features that occur in both sets of spectra. A comparison is made with the conventional ordinary least squares (OLS) approach. Both methods (OLS and ASLS) are illustrated with simulations and are tested for size-exclusion chromatography with infrared detection (SEC-IR) of mixtures of polymer standards. Both methods are able to provide information about new spectral features. It is shown that the ASLS-based procedure yields the best recovery of new features in the simulations and in the SEC-IR experiments. Band positions and band shapes of new spectral features are better retrieved with the ASLS than with the OLS method, even those which could hardly be detected visually. Depending on the spectroscopic technique used, the ASLS-based method facilitates identification of the new chemical compounds. © 2005 American Chemical Society.

This paper presents a first structural characterization of isolated patatin, the major potato tuber protein, at ambient and elevated temperatures. Isolated patatin at room temperature is a highly structured molecule at both secondary and tertiary levels. It is estimated from far-ultraviolet circular dichroism data that about 33% of the residues adopts an α-helical and 46% a β-stranded structure. Patatin is thermally destabilized at temperatures exceeding 28°C, as was indicated by near-ultraviolet circular dichroism. It was shown that parts of the α-helical contributions unfold in the 45-55°C region, whereas the β-stranded parts unfold more gradually at temperatures of 50-90°C. This was confirmed with Fourier-transform infrared spectroscopy. Differential scanning calorimetry indicated a cooperative transition between 50-60°C, most likely reflecting the unfolding of α-helical parts of the molecule. Furthermore, fluorescence spectroscopy confirmed a global unfolding of the protein between 45-55°C. The observed unfolding of the protein coincides with the inactivation of the patatin enzyme activity and with the precipitation as occurs in the potato fruit juice upon heating. At high temperatures, patatin still contains some helical and stranded structures. Upon cooling the protein partly refolds, it was observed that mainly α-helical structures were formed.

A reversed-phase liquid chromatography-linear ion trap-Fourier transform ion cyclotron resonance-mass spectrometry method was developed for the profiling of lipids in human and mouse plasma. With the use of a fused-core C₈ column and a binary gradient, more than 160 lipids belonging to eight different classes were detected in a single LC-MS run. The method was fully validated and the analytical characteristics such as linearity (R², 0.994-1.000), limit of detection (0.08-1.28 μ/mL plasma), repeatability (RSD, 2.7-7.9%) and intermediate precision (RSD, 2.7-15.6%) were satisfactory-The method was successfully applied to p53 mutant mice plasma for studying some phenotypic effects of p53 expression. © 2008 American Chemical Society.

This review summarises the most recent developments in ginseng analysis, in particular the novel approaches in sample pre-treatment and the use of high-performance liquid-chromatography-mass spectrometry. The review also presents novel data on analysing ginseng extracts by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry (Fourier transform mass spectrometry)in the context of metabolomics. Copyright © 2007 John Wiley & Sons, Ltd.

In this study the relation between the ability of protein self-association and the surface properties at air-water interfaces is investigated using a combination of spectroscopic techniques. Three forms of chicken egg ovalbumin were obtained with different self-associating behavior: native ovalbumin, heat-treated ovalbumin-being a cluster of 12-16 predominantly noncovalently bound proteins, and succinylated ovalbumin, as a form with diminished aggregation properties due to increased electrostatic repulsion. While the bulk diffusion of aggregated protein is clearly slower compared to monomeric protein, the efficiency of transport to the interface is increased, just like the efficiency of sticking to rather than bouncing from the interface. On a timescale of hours, the aggregated protein dissociates and adopts a conformation comparable to that of native protein adsorbed to the interface. The exerted surface pressure is higher for aggregated material, most probably because the deformability of the particle is smaller. Aggregated protein has a lower ability to desorb from the interface upon compression of the surface layer, resulting in a steadily increasing surface pressure upon reducing the available area for the surface layer. This observation is opposite to what is observed for succinylated protein that may desorb more easily and thereby suppresses the buildup of a surface pressure. Generally, this work demonstrates that modulating the ability of proteins to self-associate offers a tool to control the rheological properties of interfaces.

The on-line coupling of comprehensive two-dimensional liquid chromatography (liquid chromatography × size-exclusion chromatography, LC × SEC) and infrared (IR) spectroscopy has been realized by means of an IR flow cell. The system has been assessed by the functional-group analysis of a series of styrene-methylacrylate (SMA) copolymers with varying styrene content. Ultraviolet (UV) detection was used as a detection technique to verify the detection with IR. The LC × SEC-IR functional-group contour plots (comprehensive chromatograms) obtained for styrene were in agreement with the contour plots constructed from the UV signal. In addition, contour plots can be obtained from non-UV-active groups. One such plot, for the carbonyl-stretching vibration of methylacrylate (MA), is shown. Selective detection of MA proved possible using flow cell IR detection. The combination of the contour plots for styrene and MA allowed a full characterization of the copolymer and it was revealed that the present series of SMA copolymers exhibited homogeneous chemical-composition distributions (CCDs). In addition, commercially available fast-SEC columns have been assessed in this study with respect to their potential to serve as second-dimension separation columns. © 2005 Elsevier B.V. All rights reserved.

Models of P-type ATPase predict that membrane-embedded fragments represent about 20% of the protein and adopt an all-α-helical structure. While this prediction was confirmed for the Ca2+ -ATPase [Corbalan-Garcia, S., Teruel, J., Villalain, J. and Gomez-Fernandez, J. (1994) Biochemistry 33, 8247-8254], it is at odds with recent experimental evidence gathered on the Neurospora crassa plasma membrane H+ -ATPase [Vigneron, L., Ruysschaert, J.-M. and Goormaghtigh, E. (1995) J. Biol. Chem. 270, 17685-17696] and on the gastric H+,K+ -ATPase [Raussens, V., Ruysschaert, J.-M. and Goormaghtigh, E. (1997) J. Biol. Chem. 276, 262-270]. Extensive proteinase K proteolysis of open gastric tubulovesicles was performed here to generate the membrane-protected fragments of the H+,K+ -ATPase. Secondary structure of the intact and of the membrane-protected segments was compared for oriented membrane films by attenuated total-reflection Fourier-transform infrared spectroscopy and by circular dichroism and for vesicles suspension by circular dichroism and Raman spectroscopy. All the spectroscopic data indicate that the protease-resistant membrane-bound residue of the H+,K+ -ATPase contains significant amount of β-sheet structure, both on films and in membrane suspensions. Polarized attenuated total-reflection infrared spectroscopy indicates that only the α-helical content of protease-resistant membrane-bound residue of the H+,K+ -ATPase is oriented (parallel) with respect to the membrane normal. Raman spectroscopy reveals that Phe residues are preferentially removed by protease activity. Evaluation of the amount of removed Phe and Tyr residues places constraints on the model of membrane insertion of the H+,K+ -ATPase.