Applied Spectroscopy

Ligand binding alters the conformational structure and physico-chemical characteristics of bovine folate binding protein (FBP). For the purpose of achieving further information we analyzed ligand (folate and methotrexate) -induced changes in the fluorescence landscape of FBP. Fluorescence excitation and emission two-dimensional (2D) spectra were recorded over a wide range of wavelengths on a Perkin-Elmer LS 55 spectrofluorometer at varying pH in different buffers, and the resulting three-dimensional data were subjected to a chemometric analysis, parallel factor analysis (PARAFAC). The most important finding was the occurrence of two maximum intensity emission wavelengths of tryptophan, 350 nm (component one) and 330 nm (component two). In contrast to the first component, the score of the short wavelength component increased with increasing ligation of FBP. Since the emission wavelengths of indole groups in tryptophan shorten with increasing distance from the solvent surface of proteins, an increasing number of the 11 tryptophan residues seem to reorientate from the solvent surface to the interior of FBP with increasing ligation. The sharp decrease in hydrophobicity at pI=7–8 following binding of folate accords fairly well with the disappearance of strongly hydrophobic tryptophan residues from the solvent-exposed surface of FBP. The PARAFAC has thus proven useful to establish a hitherto unexplained link between parallel changes in conformational structure and physico-chemical characteristics of FBP induced by folate binding. Parameters for ligand binding derived from PARAFAC analysis of the fluorescence data were qualitatively and quantitatively similar to those obtained from binding of radiofolate to FBP. Herein, methotrexate exhibited a higher affinity for FBP than in competition with radiofolate. This could suggest a rapid and firm complexation of folate to FBP, blocking access of competing ligands.

The variations of the fluorescence emission of carmine lake travelling through an absorbing and scattering medium, such as a paint layer, were investigated by ultraviolet (UV)–visible absorption, fluorescence spectroscopy, and imaging techniques. Samples of the lake were studied in dilute and saturated solutions, on a reference test panel and a real case study. Relevant spectral modifications have been observed as a function of the lake concentration mainly consisting of a fluorescence quenching, red shift of emission maxima, and deformation of emission band. The application of a correction factor based on the Kubelka–Munk model allowed fluorescence spectra obtained in solution and on painted samples of known composition to be compared and correlated, highlighting that the fluorescence of the lake within paint layers is affected by both self-absorption and aggregation phenomena. This approach has been successfully applied on a painting by G. Vasari for the noninvasive identification of carmine lake. The results reported here emphasize the necessity of taking physical phenomena into account in the interpretation of the fluorescence spectra for a proper and reliable characterization and identification of painting materials in works of art.

By utilizing ultraviolet (UV)-visible absorption spectroscopy, circular dichroism (CD), resonance light scattering (RLS), and transmission electron microscope (TEM) techniques, the toxic interaction of methyl violet (MV) with calf thymus DNA (ctDNA) was investigated at the molecular level. The UV-visible absorption spectra results showed that MV intercalated into ctDNA base pairs at low MV concentrations, while MV was attached to the negative charged phosphate groups at higher concentrations and the binding constant (K^θ_25°C) was 1.80 × 10³ L mol⁻¹. The absorbency results showed that the genotoxic interaction of MV with ctDNA is under the electrostatic binding mode. MV had strong toxic interaction with ctDNA at 4.0 × 10⁻⁵ mol L⁻¹ MV and pH 6.5. The toxic interaction of MV with ctDNA can result in the formation of massive aggregates and change of the ctDNA conformation, which showed a significant linear dose-response relationship.

A molecular spectral imaging system has been developed based on microscopy and spectral imaging technology. The system is capable of acquiring molecular spectral images from 400 nm to 800 nm with 2 nm wavelength increments. The basic principles, instrumental systems, and system calibration method as well as its applications for the calculation of the stain-uptake by tissues are introduced. As a case study, the system is used for determining the pathogenesis of diabetic retinopathy and evaluating the therapeutic effects of erythropoietin. Some molecular spectral images of retinal sections of normal, diabetic, and treated rats were collected and analyzed. The typical transmittance curves of positive spots stained for albumin and advanced glycation end products are retrieved from molecular spectral data with the spectral response calibration algorithm. To explore and evaluate the protective effect of erythropoietin (EPO) on retinal albumin leakage of streptozotocin-induced diabetic rats, an algorithm based on Beer–Lambert's law is presented. The algorithm can assess the uptake by histologic retinal sections of stains used in quantitative pathology to label albumin leakage and advanced glycation end products formation. Experimental results show that the system is helpful for the ophthalmologist to reveal the pathogenesis of diabetic retinopathy and explore the protective effect of erythropoietin on retinal cells of diabetic rats. It also highlights the potential of molecular spectral imaging technology to provide more effective and reliable diagnostic criteria in pathology.

The intercalation of molten polymer into layered silicates (nanoclays) is a critical step in the fabrication of commercial polymer nanocomposite products, so it is therefore very important to understand the process of intercalation and exfoliation, as well as to properly characterize the final state achieved. Following on our recent work demonstrating the potential of transmission infrared spectroscopy for this purpose, we show in this work how the attenuated total reflection technique can be used to study the intercalation process in real time. Although various aspects of the Si–O stretching bands near 1150–950 cm⁻¹ are sensitive to the intercalation, the most sensitive parameter is the relative intensity of the out-of-plane band near 1077 cm⁻¹. A method based on the second derivative of the spectrum has been developed to quantify this aspect. The application of this approach is described in detail and examples are given involving different polymers.

In the present study, the orientation of a poly(3-hydroxybutyrate) (PHB)/poly(ε-caprolactone) (PCL) blend was monitored during uniaxial elongation by rheo-optical Fourier transform infrared (FT-IR) spectroscopy and analyzed by generalized two-dimensional correlation spectroscopy (2D-COS). The dichroism of the δ(CH₂) absorption bands of PHB and PCL was employed to determine the polymer chain orientation in the PHB/PCL blend during the elongation up to 267% strain. From the PHB and PCL specific orientation functions it was derived that the PCL chains orient into the drawing direction while the PHB chains orient predominantly perpendicular to the applied strain. To extract more detailed information about the polymer orientation during uniaxial elongation, 2D-COS analysis was employed for the dichroic difference of the polarization spectra recorded during the drawing process. In the corresponding synchronous and asynchronous 2D correlation plots, absorption bands characteristic of the crystalline and amorphous regions of PHB and PCL were separated. Furthermore, the 2D-COS analysis revealed that during the mechanical treatment the PCL domains orient before the PHB domains.

The hydrogen bonding properties of a representative molecule, 2-mercaptoethanol (ME), of which two functional groups OH and SH are believed to interact competitively or selectively with proton-accepting molecules, have been studied. Three binary systems, namely ME–CCl₄, ME–dimethyl sulfoxide (DMSO), and ME–acetone, were investigated with excess infrared absorption spectroscopy. It is found that when DMSO or acetone is added into ME, they preferentially form hydrogen bonds with OH, and the hydrogen bonds in the ME–DMSO system are stronger than those in the ME–acetone system. When CCl₄ is added into ME, the weak hydrogen bonds involving the SH group are broken preferentially with increasing CCl₄ concentration. The dissociation process of ME in the inert diluent CCl₄ over the entire concentration range has been discussed in detail. In the very low concentration range of CCl₄, the highly hydrogen bonded ME multimers mainly break into medium-sized aggregates. The amount of the trimers and dimers first increases and then, at x(CCl₄) = 0.77, begins to decrease. These results suggest that excess infrared spectroscopy can provide detailed molecular pictures in liquid solutions containing complex hydrogen bonding interactions. It can also help to locate individual peak positions in the deconvolution of overlapped absorption bands.

This paper describes a partial least squares (PLS) based automatic procedure to correct for changes in the spectral contribution of the solvent or solvent mixtures from solute spectra recorded in these solvents. The procedure was developed for successful on-line Fourier transform infrared (FT-IR) detection in gradient high-performance liquid chromatography (HPLC) separations. It requires a reference FT-IR data set containing all possible combinations of the expected variation in solvent composition. Furthermore, a spectral region (A) in these spectra is required where the solvents show absorption but the analytes do not. This is the case for the system ACN:H₂O, an often-applied solvent mixture in gradient HPLC, in the spectral region from 2300–2400 cm⁻¹. By focusing on (A) the developed numerical method selects an appropriate background spectrum from the reference data set, which is then subtracted from the analyte spectra. The method was programmed in Matlab and tested on different isocratic and gradient on-line reversed phase liquid chromatography–Fourier transform infrared spectrometry (LC-FT-IR) data sets. This work describes a successful method to perform eluent correction in on-line coupling of FT-IR spectrometry with gradient LC.

Wavelet transforms are an extremely powerful tool when it comes to processing signals that have very “low frequency” components or non-periodic events. Our particular interest here is in the ability of wavelet transforms to remove backgrounds of spectroscopic signals. We will discuss the case of surface-enhanced Raman spectroscopy (SERS) for illustration, but the situation it depicts is widespread throughout a myriad of different types of spectroscopies (IR, NMR, etc.). We outline a purpose-built algorithm that we have developed to perform an iterative wavelet transform. In this algorithm, the effect of the signal peaks above the background is reduced after each iteration until the fit converges close to the real background. Experimental examples of two different SERS applications are given: one involving broad backgrounds (that do not vary much among spectra), and another that involves single molecule SERS (SM-SERS) measurements with narrower (and varying) backgrounds. In both cases, we will show that wavelet transforms can be used to fit the background with a great deal of accuracy, thus providing the framework for automatic background removal of large sets of data (typically obtained in time-series or spatial mappings). A MATLAB® based application that utilizes the iterative algorithm developed here is freely available to download from http://www.victoria.ac.nz/raman/publis/codes/cobra.aspx.

Raman spectroscopy is used to investigate the α- and β-crystalline polymorphs of isotactic polypropylene. Raman spectra of the polymorphs show some minor differences, in particular the wavenumber shifting of certain scattering bands, which reflect the environment of an isolated macromolecular chain within the packing unit cell of the different crystal forms. For example, the shifting of the 842 cm⁻¹ scattering band, which is the most susceptible band to cause intermolecular interactions, is used to generate a structural map of one β-spherulite among α-spherulites with a well-defined resolution. Anisotropy of the crystalline phase orientation within a β-spherulite is also investigated by performing polarized Raman spectroscopy measurements. Differences in the polarized spectra are then finely exploited to map the radial lamellae orientation distribution within one β-spherulite.

The diffusion of molecular species within a sulfuric acid–water system has been monitored by Raman spectroscopy, a thermodynamic-chemical model of the mass transport properties of the species has been established, and its parameters optimized. It has been shown that the non-ideality of this multicomponent system plays a crucial role in its mass transport properties, which have been explained in terms of a diffusion model for the molecular species. The individual effective diffusion coefficients are not constant (characteristic of ideal systems) but are a function of the concentration of the species in solution. The model has been conceived in such a way that it can be adapted to any multicomponent mixture when the equilibriums among the ions are known. Raman spectroscopy provides the means to derive the speciation and concentration of species in multicomponent systems, and we have shown that the model-based measurement of the diffusion properties using Raman is a robust and accurate technique that allows for measuring the individual diffusion coefficients of the species in the mentioned system.

In this paper we demonstrate how broadband cavity enhanced absorption spectroscopy (CEAS) with supercontinuum (SC) radiation in the near-infrared spectral range can be used as a sensitive, multiplexed, and simple tool to probe gas-phase species in high-temperature environments. Near-infrared SC radiation is generated by pumping a standard single-mode fiber with a picosecond fiber laser. Standard low reflectivity mirrors are used for the cavity and an optical spectrum analyzer is used for the detection of gas-phase species in combustion. The method is demonstrated by measuring flame generated H₂O in the 1500 to 1550 nm region and room-temperature CO₂ between 1520 nm and 1660 nm. The broadband nature of the technique permits hundreds of rotational features to be recorded, giving good potential to unravel complex, convoluted spectra. We discuss practical issues concerning the implementation of the technique and present a straightforward method for calibration of the CEAS system via a cavity ringdown measurement. Despite the large spectral variation of SC radiation from pulse to pulse, it is shown that SC sources can offer good stability for CEAS where a large number of SC pulses are typically averaged.

In view of the considerable number of analyses necessary in phytoremediation and plant biology studies, it is important that the analytical procedures used for elemental determination in plant tissues should be fast and cheap, with simple sample preparation, and of adequate accuracy and precision. The aim of the present work was focused on the possibilities and drawbacks of a low-cost benchtop energy dispersive X-ray fluorescence (EDXRF) instrument to be used as an analytical technique for studying the potential use of sunflowers (Helianthus annuus) for the phytoremediation of an abandoned Pb/Zn mining area. The simplicity and the ability to operate at room temperature and open air conditions provides a flexible setup for the rapid analysis of vegetation material without complicated previous sample pretreatments. With the help of different collimator caps, a modest lateral resolution (from 200 μm to 1 mm) was achieved and it was thought to be small enough to study the accumulation of Pb and Zn and its distribution between parts of the vegetation specimens (roots, stems, and leaves); the limits of detection achieved (0.6 ng for Zn and 3.0 ng for Pb) proved to be suitable for the intended purpose. Therefore, obtained data revealed that EDXRF spectrometry could be a useful tool to better understand the metal uptake, translocation, and tolerance mechanisms in vegetation species related to biomonitoring and phytoremediation studies.