Applied Spectroscopy

The analysis of body fluid traces during forensic investigations is a critical step in determining the key details of a crime. Several confirmatory and presumptive biochemical tests are currently utilized. However, these tests are all destructive, and no single method can be used to analyze all body fluids. This review outlines recent progress in the development of a novel universal approach for the nondestructive, confirmatory identification of body fluid traces using Raman spectroscopy. The method is based on the use of multidimensional spectroscopic signatures of body fluids and accounts for the intrinsic heterogeneity of dry traces and donor variation. The results presented here demonstrate that Raman spectroscopy has potential for identifying traces of semen, blood, saliva, sweat, and vaginal fluid with high confidence.

Important interfacial processes in disciplines ranging from medicine to the separations sciences occur over a wide range of pressures, temperatures, and time scales. In this paper we report a new high-pressure total internal reflection fluorescence (HP-TIRF) apparatus that allows rapid fluorescence measurements of sub-monolayers in contact with liquids and supercritical fluids between 293 K and 353 K and up to 250 bar with picosecond time resolution. We use the HP-TIRF system to study the in-plane rotational reorientation dynamics of the fluorescent probe BODIPY 494/503 (C_2v symmetry) covalently attached to silica surfaces that have been silanized with n-propyltrimethoxysilane (C₃-TMOS) or 3,3,3-trifluoropropyltrimethoxysilane (CF₃-TMOS) when the interface is subjected to pure supercritical carbon dioxide (scCO₂). The in-plane BODIPY 494/503 rotational reorientation dynamics are assessed by using the Debye–Stokes–Einstein expression. As the scCO₂ density increases the local microviscosity surrounding the tethered BODIPY 494/ 503 molecule decreases. The terminal group (CH₃ versus CF₃) within the silane monolayer governs the onset and absolute magnitude of the observed viscosity changes. The results are explained in terms of the well-known solubility of fluorine-containing species in scCO₂.

The application of fluorescence excitation–emission matrix (EEM) spectroscopy to the quantitative analysis of complex, aqueous solutions of cell culture media components was investigated. These components, yeastolate, phytone, recombinant human insulin, eRDF basal medium, and four different chemically defined (CD) media, are used for the formulation of basal and feed media employed in the production of recombinant proteins using a Chinese Hamster Ovary (CHO) cell based process. The comprehensive analysis (either identification or quality assessment) of these materials using chromatographic methods is time consuming and expensive and is not suitable for high-throughput quality control. The use of EEM in conjunction with multiway chemometric methods provided a rapid, nondestructive analytical method suitable for the screening of large numbers of samples. Here we used multiway robust principal component analysis (MROBPCA) in conjunction with n-way partial least squares discriminant analysis (NPLS-DA) to develop a robust routine for both the identification and quality evaluation of these important cell culture materials. These methods are applicable to a wide range of complex mixtures because they do not rely on any predetermined compositional or property information, thus making them potentially very useful for sample handling, tracking, and quality assessment in biopharmaceutical industries.

The interaction of bisphenol A with bovine hemoglobin (BHb) under physiological conditions was investigated by using fluorescence, ultraviolet-visible (UV-Vis) absorption, circular dichroism (CD), and molecular modeling. The experimental results showed that BPA can bind with BHb to form a complex. The binding constant Ka and the number of binding sites n were calculated to be 1.49 × 10⁵ L mol⁻¹ and 1, respectively. Molecular modeling study revealed that BPA bound into BHb central cavity, and the binding mode of BPA-BHb complex could be hydrogen bonding. The UV-Vis absorption and CD spectra indicated that the secondary structure of BHb was altered, which may affect physiological functions of hemoglobin. This work is helpful for clarifying the molecular toxic mechanism of BPA in vivo.

Sum frequency generation (SFG) microscopy images of cotton cellulose fibers were observed at the infrared wavenumber of ∼ 2945 cm⁻¹ and with a spatial resolution of 2 μm. Domains of different cellulose microfibril bunches were observed and they showed different second-order nonlinear responses. The intensity of the peak of the asymmetric CH₂ stretching mode at 2945 cm⁻¹ depended strongly on the orientation of the electric fields of the incident visible and infrared light with respect to the cellulose fiber axis. The second-order nonlinear susceptibility arising from the chirality in the cellulose structure was found to be dominant. The SFG of the cross section of the cellulose fiber was relatively weak and showed a different spectrum from that measured from the side of the fiber axis.

A compact rack-mounted cavity ring-down spectrometer (CRDS) for simultaneous measurements of the nocturnal nitrogen oxides NO₃ and N₂O₅ in ambient air is described. The instrument uses a red diode laser to quantify mixing ratios of NO₃ (at its absorption maximum at 662 nm) and of N₂O₅ following its thermal dissociation to NO₃ in a second detection channel. The spectrometer is equipped with an automated zeroing and calibration setup to determine effective NO₃ absorption cross-sections and NO₃ and N₂O₅ inlet transmission efficiencies. The instrument response was calibrated using simultaneous measurements of NO₂, generated by thermal dissociation of N₂O₅ and/or by titration of NO₃ with excess NO, using blue diode laser CRDS at 405 nm. When measuring ambient air, the (2σ, 10 s) precision of the red diode CRDS varied between 5 and 8 parts-per-trillion by volume (pptv), which sufficed to quantify N₂O₅ concentrations under moderately polluted conditions. Sample N₂O₅ measurements made on a rooftop on the University of Calgary campus in August 2010 are presented. A maximum N₂O₅ mixing ratio of 130 pptv was observed, corresponding to a steady-state lifetime of less than 50 min. The NO₃ mixing ratios were below the detection limit, consistent with their predicted values based on equilibrium calculations. During the measurement period, the instrument response for N₂O₅ was 70% of the theoretical maximum, rationalized by a slight mismatch of the laser diode output with the NO₃ absorption line and a N₂O₅ inlet transmission efficiency less than unity. Advantages and limitations of the instrument's compact design are discussed.

The development of a hollow core waveguide (HWG) gas sensor in combination with a fast and compact near-infrared (NIR) spectrometer is presented. The spectrometer operates in the spectral range of 1200–1400 nm and may thus be applied for the detection of gas-phase analytes providing NIR absorptions in that spectral window such as, e.g., methane. Since mid-infrared spectroscopy in combination with HWGs has already been successfully demonstrated for probing hydrocarbons in the gas phase, the present study investigates the achievable sensitivity in the NIR spectral regime. Methane has been selected as an exemplary analyte due to the fact that it shows strong absorption features in the mid-infrared (mid-IR) fingerprint area, but also overtone bands in the NIR. Since the HWG simultaneously serves as a miniaturized absorption gas cell and as an optical waveguide for NIR radiation, a compact yet optical and cost-efficient sensor device was established providing an interesting alternative in target sensing for mid-IR devices. The achieved limit of detection (LOD) was 5.7% (vol./vol.) methane for a 9.5 cm long HWG, 1.6% (vol./ vol.) methane for a 39.1 cm long HWG, and 1.3% (vol./vol.) methane for a setup using a 77.4 cm long HWG, which provides the most practical HWG dimensions among the three investigated setups. Limit of quantitation (LOQ) values were calculated at 20.1% (vol./vol.) methane, 8.7% (vol./ vol.) methane, and 5.6% (vol./vol.) methane, respectively.

This paper reports simultaneous photoinduced precipitation-based online preconcentration of target analytes at the inner walls in capillary zone electrophoresis (CZE) and surface-enhanced near-field crossed-beam photothermal-lens detection of the preconcentrated analytes. A simple technique using online readjustment of the optical scheme of the thermal-lens detector in the course of the separation for gaining optimum sensitivity for both water-soluble and precipitated analytes is proposed. It provides a considerable decrease in the limits of detection (LOD) with good concordance with the previously developed theoretical approach to this combination (D. A. Nedosekin, W. Faubel, M. A. Proskurnin, and U. Pyell, Talanta, 78, 682–690 (2009)). As a result, an enhancement of more than an order of magnitude in the limit of detection of the photoactive 4-aminoazobenzene compared to conventional thermal-lens detection in CZE is achieved while retaining very good sensitivity for unabsorbed analyte (Mordant Yellow 7). The application of the thermal-lens detector to the investigation of laser-induced reactions in flow in capillaries is discussed.

Raman spectroscopy and X-ray fluorescence (XRF) spectroscopy are often used as complementary techniques that are well suited for the analysis of art objects because both techniques are fast, sensitive, and noninvasive and measurements can take place in situ. In most of these studies, both techniques are used separately, in the sense that the spectra are evaluated independently and single conclusions are obtained, considering both results. This paper presents a data fusion procedure for Raman and XRF data for the characterization of pigments used in porcelain cards. For the classification of the analyzed points of the porcelain cards principal component analysis (PCA) was used. A first attempt was made to develop a new procedure for the identification of the pigments using a database containing the fused Raman–XRF data of 24 reference pigments. The results show that the classification based on the fused Raman–XRF data is significantly better than the classifications based on the Raman data or the XRF data separately.

A pharmaceutical formulation containing metformin hydrochloride (MET), hydroxypropyl cellulose (HPC), and microcrystalline cellulose (MCC) was wet granulated with varying amounts of water and the structure of the obtained granules was characterized by Raman chemical mapping. Univariate Raman mapping was found to be satisfactory for producing the images of the two components of interest (HPC and MCC). In addition to the images, the average Raman spectra from the maps as well as the micro-Raman spectra from the hot pixels were analyzed. HPC is found to strongly respond to the addition of water, with its domain dissipating and Raman bands becoming weaker as the water addition increases. MCC is also responsive to water, reacting similarly to HPC but to a much smaller extent and only for the largest amounts of water. Granules made with increasing water content also have improved tabletting properties and flow.

Raman spectral analysis integrated with multivariate calibration is a fast and effective solution to monitor chemical product properties. However, Raman instruments utilizing charge-coupled device (CCD) detectors suffer from occasional spikes caused by cosmic rays. Cosmic spikes can disturb or even destroy the meaningful chemical information expressed by normal Raman spectra. In online monitoring, some cosmic spikes have intensity and bandwidth similar to normal Raman peaks of chemical components when a low resolution and cost-effective Raman instrument is used. Moreover, the online Raman spectra always contain variations of strong Raman peaks and fluorescence. Current spike-removal methods seem to have difficulty detecting and recovering cosmic spikes in these online Raman spectra. Therefore, an improved algorithm is proposed. In this algorithm, a new scheme composed of intensity identification and local moving window correlation analysis is introduced for cosmic spike detection; intensity identification based on derivative spectra and local linear fitting approximation are used for the recovery of cosmic spikes. The algorithm is proved to be simple and effective and has been applied in an online Raman instrument installed at a continuous catalytic reforming unit in a refinery.

Infrared (IR) spectroscopy is widely used for studies of temperature dependent properties of liquids and solutions, such as thermal denaturation of proteins and other molecules of biological interest. The variation of the spectroscopic signals with temperature can be affected by the changes in the optical path length due to the thermal expansion of the components of the sample cell. In this report we investigate the temperature dependence of the optical path length for a liquid IR sample cell of a design typical for aqueous solution experiments. The path lengths were measured from the interference fringes, both in dry cells and with cells partially filled with water. We found that the optical path length variations are significant, on the order of several percent within the temperature range used (0–87 °C). Several commercially available spacers (Teflon, mylar, and lead) and gaskets (Teflon, lead, silicone rubber, Viton, and neoprene) were tested to find materials with either the smallest or most reproducible effect. Teflon, due to its phase transition (known as the “knee point”) near room temperature, leads to abrupt changes in path length when used as either spacer or gasket component. On the other hand, Teflon is preferred for its inertness, while several of the other tested materials, most notably lead, are not practically usable due to adhesion to the cell windows upon heating and contact with the aqueous sample. The combination that yielded the most reproducible results, with minimal complications due to adhesion, was Teflon spacer with neoprene gaskets. The implications of the optical path length changes for the temperature-dependent IR experiments and their possible corrections are discussed.

In order to determine the bulk optical properties of a Bacillus subtilis culture during growth phase we investigated the effect of sample thickness on measurements taken with different measurement configurations, namely total diffuse reflectance and total diffuse transmittance. The bulk optical properties were extracted by inverting the measurements using the radiative transfer theory. While the relationship between reflectance and biomass changes with sample thickness and the intensity (absorbance) levels vary significantly for both reflectance and transmittance measurements, the extracted optical properties show consistent behavior in terms of both the relationship with biomass and magnitude. This observation indicates the potential of bulk optical properties for building models that could be more easily transferable compared to those built using raw measurements.

A simple phosphoroscope with no moving parts is described. In one scan the total luminescence, the long-lived phosphorescence, and the short-lived fluorescence can be determined. A 50% duty cycle excitation from a diode laser is used to excite the sample, and from the digitized waveform the phosphorescence is extracted from the off period, the total emission from the full cycle, and the fluorescence from the on period corrected for the phosphorescence contribution. The performance of the system is demonstrated using room-temperature phosphorescence of organic dyes in boric acid glasses, a multi-emissive boron-polymer dye, and a europium chelate.