Applied Spectroscopy

For phase-separated multicomponent polymeric systems, characterization of the interface between the components is particularly challenging. We have observed an optical effect in the infrared that can be used to image the interface specifically. This method yields images of the interfaces based on the interfaces showing apparent absorption arising from changes in refractive index at frequencies far from the specific frequencies associated with the components of the mixture. This method has been applied to multicomponent samples of polymer-dispersed liquid crystals where the nature of the interface can be specifically altered by the application of an electric potential across the sample. Effects of this optical phenomenon on spectra from such multicomponent systems are discussed, and factors that complicate quantitative analysis of data from interfacial regions have been pointed out.

The authentication of food is a very important issue both for the consumers and for the food industry with respect to all levels of the food chain from raw materials to finished products. Corn starch can be used in a wide variety of food preparation as bakery cream fillings, sauce, or dry mixes. There are many modifications of the corn starch in connection with its use in the agrofood industry. This paper describes a novel approach to the classification of modified starches and the recognition of their modifications by artificial neural network (ANN) processing of attenuated total reflection Fourier transform spectroscopy (ATR/FT-IR) spectra. Using the self-organizing artificial neural network of the Kohonen type, we can obtain natural groupings of similarly modified samples on a two-dimensional plane. Such mapping provides the expert with the possibility of analyzing the distribution of samples and predicting modifications of unknown samples by using their relative position with respect to existing clusters. On the basis of the available information in the infrared spectra, a feedforward artificial neural network, trained with the intensities of the derivative infrared spectra as input and the starch modifications as output, allows the user to identify modified starches presented as prediction samples.

A combined Raman microprobe and laser trapping system using near-infrared (NIR) laser light was developed for the investigation of single organic microdroplets. The NIR laser light is noninvasive and reduces fluorescence interference in the Raman spectrum for organic molecules. The focused laser beam used for the laser trapping of a microdroplet serves simultaneously as the laser microprobe for Raman measurement. With this system, the focused laser spot is about 1 μm in diameter, which is small enough for the laser trapping of a single toluene microdroplet in water. The system also makes it possible to visualize a focused laser spot together with a laser-trapped microdroplet by using holographic notch filters. The Raman spectrum for a single laser-trapped toluene microdroplet can be obtained from below 100 cm⁻¹ to above 3000 cm⁻¹ with a charge-coupled device (CCD) detector. Fluorescence interference in the Raman spectrum is completely removed by using NIR laser light. The signal-to-noise ratio (SNR), defined as the ratio of the peak height to the standard deviation of the baseline noise in the spectrum, exceeded 250 for the 1003 cm⁻¹ band of a toluene microdroplet at 1 s, which is sufficient to allow identification of the molecular species of a microdroplet.

A method for simultaneous two-dimensional visualization of water in both gas and liquid phase is presented. This laser-based diagnostic technique uses a combination of two-photon laser-induced fluorescence (LIF) and spontaneous Raman scattering. A tunable KrF excimer laser, operating near 248 nm, was used as an excitation source. The technique was demonstrated on single water droplets and their surrounding gas-phase content. Prior to the visualization experiments, spectroscopic measurements were performed to find optimum filtering conditions.

A confocal direct imaging Raman microscope (CDIRM) based on two synchronized scanning mirrors, a monochromator, and two charge-coupled device (CCD) cameras has been developed. With this system it is possible to make both Raman spectra of a small measurement volume and images of a larger sample area in one specific Raman band. The spatial resolution of the system was determined for two limiting situations: a small sphere and a thin layer. The image of a 0.282 μm sphere appeared to have a full width at half-maximum (FWHM) of 1.2 μm in the axial and 0.37 μm in the lateral direction, whereas the image of a 275 nm layer showed an FWHM of 1.4 μm in the axial direction. Confocal Raman images were made of the DNA and protein distribution in polytene chromosomes with a relatively weak Raman signal [0.1 photons/(second·pixel)]. Further, a three-dimensional Raman image of the drug distribution in a phthalocyanine-incubated fixed cell is presented. These examples show that the CDIRM can be used to image samples with a weak Raman signal and that three-dimensional images of the distribution of specific molecules in a sample can be made.

High-modulus and high-strength carbon fibers were submitted to oxidation plus sizing and grinding treatments. Subsequent changes at the fiber surfaces were studied by Raman spectrometry. X-ray diffractograms and Raman spectra of the fresh fibers were compared with those of other carbon materials with different graphitization degrees. The industrial oxidation +sizing treatment induces disorder at the fibers surface, this being of a higher order of magnitude than that introduced by grinding. However, this disorder is different in nature from the intrinsic disorder typical for little graphitized carbon materials.

The surface segregation of a series of two-component polymer blends of polystyrene/poly(dimethylsiloxane)-co-polystyrene has been studied by attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy. We examined the details of calibration for quantitative ATR FT-IR. The quantitation is based on calibration of Beer's law by transmission FT-IR measurements. The effect of spectral distortion and penetration depth dependence of radiation wavelength due to ATR sampling is evaluated. ATR spectra are corrected for penetration depth dependence before being used for quantitation. The corrected ATR spectra were compared with the transmission spectra of the same samples. Peak area ratios using one peak from each component were measured for the determination of the concentration of surface poly(dimethylsiloxane). The correction and calibration procedure improves the accuracy of quantitative surface analysis in the range of micrometer sampling depth. Unlike other approaches for quantification of ATR data on polymers, this method can be used for systems with a complex, unknown concentration gradient near the surface such as the blend system reported here.

A simplified equation for attenuated total reflection (ATR) spectral intensity in a three-phase system under a small absorption condition is derived. The spectral intensity is expressed in terms of a linear function of the bulk spectral intensities or absorption coefficients of the absorbing media. The applicability and limitation of the simplified equation are verified by comparing the spectral intensity obtained from the simplified equation to that from the exact equation.

The calculation of an absorbance spectrum depends on the measurement of a blank, or background spectrum. In many cases, such as the determination of atmospheric constituents with the use of open-path Fourier transform infrared spectroscopy (FT-IR) or the determination of water vapor in a gaseous sample, it is very difficult to obtain a good background spectrum. The difficulty is due to the fact that it is nearly impossible in these situations to measure a spectrum with no analyte features present. We present a method of generating a background spectrum based on filtering the analyte features from the sample spectrum. When the filtering method is used, the accuracy of the results obtained is found to be dependent upon the analyte peak width, peak height, and type of filter employed. Guidelines for the use of this background generation technique for quantitative determinations are presented.

Vibrationally enhanced infrared and vibrationally enhanced Raman four-wave mixing spectroscopy are experimentally explored with the use of several model systems. Studies involving a mixture of chlorobenzene, dichlorobenzene, and deuterobenzene were performed to see whether a simultaneous resonance of a combination band could enhance the Raman ring breathing mode. The results show strong interference effects that may be responsible for a suppression of the Raman resonances. Studies were also carried out in systems of chloroform/deuterobenzene and hexane/deuterobenzene. The latter samples showed intensity increases and line-shape changes that are consistent with vibrational enhancement. The discovery of these enhancements form the basis for developing infrared four-wave mixing as a method for extracting new information about molecular vibrations.

Near-infrared spectra (1300–2500 nm) collected from lysed blood solutions were shown to correlate with the pH of the solutions measured potentiometrically. Cross-validated partial least-squares (PLS) models were developed from these spectral data, which provided standard error of prediction (SEP) values below 0.05 pH units for a pH range of 1.0 (6.8–7.8). Experiments were designed to eliminate possible correlation between pH and other components in the blood in order to ensure that variations in the spectral data correlated to pH were due to hydrogen ion changes only. Further work was performed to discern the primary source of pH information in the lysed blood spectra by using spectra collected from plasma and histidine solutions. The blood, plasma, and histidine data sets were compared with the use of loading vectors from principal component analysis (PCA). These loading vectors show that variations in the spectra of the titrated amino acid histidine mimic those seen in lysed blood, but not those seen in plasma. These results suggest that histidine residues of hemoglobin are providing the spectral variation necessary for pH modeling in the lysed blood solutions. It is further shown that the observed pH-sensitive histidine bands do not arise from the exchangeable proton on the imidazole ring of histidine; rather they arise from the variation in the C–H bonds of the C2 and/or the C4 carbons of the imidazole ring as they are influenced by the titration of the nitrogen-bound proton of the imidazole ring.

Noninvasive monitoring of deep-tissue pH has been demonstrated with the use of near-infrared spectroscopic measurements and the partial least-squares (PLS) multivariate calibration technique. The near-infrared reflectance spectra (700 to 1100 nm) of the teres major muscle in five New Zealand rabbits were obtained in vivo, along with reference pH values in the muscle measured by microelectrodes. The muscle pH was varied by controlling the blood supply to the muscle. PLS analysis with cross-validation techniques, along with several data preprocessing methods, was used to relate the tissue pH to spectra. When multi-subject PLS calibration models were used to predict a new independent subject, a subject-dependent offset was observed. Several strategies for minimizing the subject-dependent offset were discussed. With a baseline subtraction procedure, the subject-dependent offset was minimized to less than 0.1 pH units while the average standard error of prediction (SEP) was close to 0.05 pH units. This result suggests that it is possible to build a single robust calibration model for all new independent subjects. Tissue chemistry during ischemia (blood flow reduction) is different from the chemistry of reperfusion (blood flow restoration), and it was found that separate calibration models permit more accurate prediction of pH.

Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved with laser-induced native fluorescence imaging microscopy. By monitoring the native fluorescence of catecholamines excited by the 275 nm laser line with an intensified charge-coupled-device (CCD) camera, we obtained good temporal and spatial resolution simultaneously without using additional fluorescent probes. Large variations were found among individual cells in terms of the amounts of catecholamines secreted and the rates of secretion. Different regions of a cell also behave differently during the secretion process. However, the degree of this local heterogeneity is smaller than in neurons and neuralgia. The influence of deep-ultraviolet (UV) laser excitation on cells is also discussed. This quantitative imaging technique provides a useful noninvasive approach for the study of dynamic cellular changes and the understanding of the molecular mechanisms of secretory processes.

Benzo[a]pyrene (B[a]P) is a potential carcinogen and mutagen, and it has been studied very extensively. In humans, B[a]P can be metabolized and becomes chemically bonded to DNA. A variety of methods are needed for the characterization of B[a]P–DNA adducts because of the structural complexity of the adducts. In this work, several solid-matrix luminescence methods were developed for the characterization of the B[a]P-DNA adducts. Both solid-matrix fluorescence and solid-matrix phosphorescence could be obtained from the adducts on Whatman No. 1 and Whatman 1PS filter paper at nanogram levels. Thallium nitrate proved to be an effective heavy atom in enhancing the solid-matrix phosphorescence of the adducts. By selective excitation of the phosphorescence of the adducts, it was possible to excite different forms of the adducts. With moisture quenching of the solid-matrix phosphorescence of the B[a]P-DNA adducts, it was feasible to selectively detect quasi-intercalated adducts. With Whatman 1PS filter paper, a unique method was developed for the solid-matrix fluorescence detection of adducts externally bound to DNA by the hydrophobic interaction of the adducts with the 1PS paper.

A multivariate optimization approach, using a central composite blocked cube-star design, has been carried out to optimize the room-temperature phosphorescence (RTP) of a ternary complex between a polycyclic aromatic hydrocarbon (acenaphthene), β-cyclodextrin, and two bromoalcohols (2-bromoethanol and 2,3-dibromopropanol). A comparative study of the effect of the two bromoalcohols in the phosphorescence response has shown that the highest RTP emission from acenaphthene included in β-CD is obtained with 2,3-dibromopropanol. An RTP determination method for acenaphthene has been proposed. Calibration graphs range between 50 and 250 ng mL⁻¹, and a detection limit of 24 ng mL⁻¹ has been established.

The NMR properties of the solid lead (II) halides and the lead (II) hydroxyhalides are reported. The analysis shows extremely wide powder patterns in all cases except the lead (II) iodide. The results indicate that one should be able to distinguish the lead (II) halide from the corresponding lead (II) hydroxyhalide in a mixture.

High-resolution (4.4 pm), time-resolved (every 0.2 s during the atomization step) spectra of indium and aluminum atoms and molecules (InF, InCl, AlF, AlCl) were obtained in a graphite tube furnace with the use of a xenon arc lamp for excitation and a linear photodiode array detector. The spectra of indium fluoride, aluminum fluoride, and aluminum chloride were shown to be composed of several vibrational bands making up the (0, 0) sequence, while the indium chloride spectrum included vibrational progressions as well. For indium fuoride and aluminum fluoride, the diatomic molecule appeared temporally earlier than atoms of the metal. Spectra of reagents used to produce indium fluoride and aluminum fluoride were similar to previous spectra in the literature that were assigned to In₂O and Al₂O, respectively. The spectral overlap of the absorption band for aluminum chloride (261.44 nm) with a lead absorption line (261.42 nm) is discussed with respect to its implications for background correction for graphite furnace atomic absorption spectrometry.

Spectrochemical analyses of aqueous solutions containing nickel or the chlorinated hydrocarbons (CHCs) C₂Cl₄, CCl₄, CHCl₃, and C₂HCl₃ were performed with the use of laser-induced breakdown spectroscopy. A Nd:YAG laser operating at 60 mJ/pulse was focused onto the surface of the liquid. Elemental line intensities were monitored in the laser-produced plume as a function of analyte concentration to determine detection limits. The limits of detection for nickel in water were 36.4 ± 5.4 mgAL and 18.0 ± 3.8 mg/L for laser irradiation at 1.06 μm and 355 nm, respectively. Ablation of pure CHCs at 355 nm produced extremely intense plasma emissions that primarily consisted of spectroscopic features attributed to CN, C₃, H, N, and Cl. The spectra were structurally identical for all the CHCs except for differences in the intensities of various emission lines. With the use of emission from neutral atomic chlorine as an identifier for CHC contamination of water, no detectable traces of these elements were observed in saturated aqueous solutions. The detection limits for the CHCs were well above the saturation limits of CHC in water.

Laser-induced breakdown spectrometry (LIBS) has been evaluated for depth profiling of phosphorus doping in silicon. Laser plasmas were formed by focusing a Nd:YAG laser (operating in the second harmonic, 532 nm) on the sample surface. Plasma emission was collected, dispersed, and detected with the use of a charge-coupled device (CCD). Experimental parameters, such as delay time and sample position relative to the laser focal point, were optimized to improve the signal-to-background ratio of phosphorus line emission. Diffusion profiles by LIBS of samples with different phosphorus diffusion steps are shown. Crater depth per pulse and ablated mass per pulse were measured to be 1.2 μm pulse⁻¹ and 50 ng pulse⁻¹, respectively. The knowledge of depth per pulse permitted the estimation of thickness of the P diffusion layer.

Spectroscopic studies of laser-induced plasma from a high-temperature superconducting material, viz., YBa₂Cu₃O₇ (YBCO), have been carried out. Electron temperature and electron density measurements were made from spectral data. The Stark broadening of emission lines was used to determine the electron density, and the ratio of line intensities was exploited for the determination of electron temperature. An initial electron temperature of 2.35 eV and electron density of 2.5 × 10¹⁷ cm⁻³ were observed. The dependence on electron temperature and density on different experimental parameters such as distance from the target, delay time after the initiation of the plasma, and laser irradiance is also discussed in detail.

Laser-induced breakdown spectroscopy has been applied to polymer samples in order to investigate the possibility of using this method for the identification of different materials. The plasma emission spectra of high-density polyethylene (HDPE), low-density polyethylene (LDPE), polyvinyl chloride (PVC), polyethylene terephthalate (PET), and polypropylene (PP) have been studied. Spectral features have been measured—for example, the 725.7 nm chlorine line, the 486.13 mm H_β line, and the 247.86 nm carbon line—whose evaluation with neural networks permits identification accuracies between 90 and 100%, depending on polymer type.

Raman shift measurements have been made on cyclohexane, by using linear interpolation between bracketing neon line positions to establish Raman band wavelengths. Factors that affect the precision of these measurements have been examined in detail. These include change of slit width, grating line spacing, neon line intensity, distance of separation of neon lines, and effects of small grating movements. All these have some effect, but the last factor is particularly important. Techniques that give standard deviations of 0.03 cm⁻¹ when measurements are made without moving the grating are degraded by about an order of magnitude when the grating is moved. This result is attributed to adventitious partitioning of signal power between adjacent detector elements. By deliberately moving the grating a small amount between otherwise replicate measurements, one can control this effect. Values for the Raman shifts for eleven cyclohexane bands, based on approximately 800 Raman spectra and an equal number of neon emission spectra, are presented. The values are compared with previous measurements. They are generally within the ±σ range of the earlier values, but the standard deviations of these results are about an order of magnitude smaller.

For the acquisition of high-quality ultraviolet resonance Raman spectra of strongly scattering samples such as membrane protein suspensions, an f/3.5 Littrow prism prefilter has been designed, built, and characterized. This prefilter has a Czerny–Turner configuration, and its focal length is 25 cm. The apex angle of the dispersive prism (20°) was chosen to provide maximum performance in the 220 to 240 nm range. The prism prefilter significantly reduced stray background due to Rayleigh scattering and visible fluorescence, while maintaining a low dispersion of 1300 cm⁻¹/mm at 253 nm as well as a large f/3.5 aperture. The sharpness of the transmission edge (at 242 nm, the T = 0% to 95% transition occurs in 1.3 nm) quantitates its effectiveness as a sharp-cut Rayleigh scattering filter. The total throughput of the prefilter is ∼60% at 235 nm and ∼50% at 632.8 nm. The utility of this prefilter is demonstrated by obtaining high signal-to-noise resonance Raman spectra of bacteriorhodopsin in a purple membrane suspension with 239.5 nm excitation.