Applied Spectroscopy

Extended multiplicative signal correction (EMSC) is used to separate and to characterize physical and chemical information in spectra from Fourier transform infrared (FT-IR) microscopy. This appears especially useful for applications in infrared spectroscopy where the scatter variance in spectra changes with the chemical variance in the sample set. In these cases the chemical information of specific bands that are assigned to functional groups is easier to interpret when the scatter information is removed from the spectra. We show that scatter (physical) information in FT-IR spectra of heat-treated beef loin is related to chemical changes due to heat treatment. This information is caused by textural changes induced by the heat treatment and expressed by physical effects as the optical path length. The chemical absorbance changes introduced in the FT-IR spectra due to heat treatment are shifts in the protein region of the infrared spectrum caused by changes in the secondary structure of the proteins. If the scatter and the chemical information is not separated properly, scatter information may erroneously be interpreted as chemical information.

Although infrared imaging is becoming more commonplace and publications demonstrating its utility are on the rise, only a small portion of the literature involves visualization strategies and techniques. In order to fully realize the potential of imaging, visualization of the data in the form of images needs to be examined more thoroughly. Visualization techniques are discussed using the dynamic process of polymer dissolution. The dissolution of poly(ethylene oxide) (PEO) was captured as a series of images showing the changes of the polymer front brought about by the dissolving solvent. Using a unique infrared band for both the polymer and solvent, each species is tracked through the entire experiment. The ability to reconstruct the dissolution from the spatially resolved infrared data proves very valuable. However, the processing of the data is not trivial and steps must be taken to ensure that the images best display the specific attributes of the species. Scaling and color mapping are demonstrated to be two ways to achieve excellent imagery.

This study developed a method to produce uniform captan surface films on a disposable nitrile glove for quantitation with a portable attenuated total reflection Fourier transform infrared (ATR-FTIR) spectrometer. A permeation test was performed using aqueous captan formulation. Uniform captan surface films were produced using solvent casting with 2-propanol and a 25 mm filter holder connected to a vacuum manifold to control solvent evaporation. The coefficient of variation of the reflectance at 1735 ± 5 cm⁻¹ was minimized by selection of the optimum solvent volume, airflow rate, and evaporation time. At room temperature, the lower to upper quantifiable limits were 0.31–20.7 μg/cm² (r = 0.9967; p ≤ 0.05) for the outer glove surface and 0.55–17.5 μg/cm² (r = 0.9409; p ≤ 0.05) for the inner surface. Relative humidity and temperature did not affect the uncoated gloves at the wavelength of captan analysis. Glove screening using ATR-FTIR was necessary as a control for between-glove variation. Captan permeation, after 8 hours exposure to an aqueous concentration of 217 mg/mL of Captan 50-WP, was detected at 0.8 ± 0.3 μg/cm² on the inner glove surface. ATR-FTIR can detect captan permeation and can determine the protectiveness of this glove in the field.

In this study, the measurement of the true vibrational circular dichroism (VCD) spectrum is considered from an experimental and theoretical approach for any general anisotropic thin solid sample exhibiting linear as well as circular birefringence (LB, CB) and dichroism (LD, CD) properties. For this purpose, we have made use of a simple model α-helix polypeptide, namely, the poly(γ-benzyl-L-glutamate) or PBLG, reference sample possessing a well-known VCD spectrum and giving rise to slightly oriented films by deposition onto a solid substrate. Also, we have used a different Fourier transform infrared modulation of polarization (PM-FTIR) optical setup with two-channel electronic processing in order to record the PM-VLD and PM-VCD spectra for various sample orientations in its film plane. All the corresponding general relations of the expected intensities in these experiments and the related properly designed calibration measurements were established using the Stokes–Mueller formalism; in addition, the residual birefringence of the optical setup and the transmittance anisotropy of the detector were estimated. From a comparative study of the results obtained in solution and in the solid state, we then propose a simple new experimental procedure to extract the true VCD spectrum of an oriented PBLG thin film: its consists of calculating the half-sum of two spectra recorded at θ and at θ ± 90° sample orientations. Moreover, the complete linear and circular birefringence and dichroism properties of the ordered PBLG thin film are estimated in the amide I and amide II vibrational regions. This allows us to establish for any sample orientation various theoretical simulations of the VCD spectra that agree nicely with the observed experimental results; this confirms that the measurement of LD and LB is in this case a prerequisite in simulating the true VCD spectrum of a partly oriented anisotropic sample. This validates our combined experimental and theoretical approach and opens the route to promising future vibrational CD studies on other macroscopic anisotropic thin film samples.

Chiral second harmonic generation (C-SHG) has been used for the label-free detection of (R)-(+)-1,1′-bi-2-naphthol (RBN) and (S)-(+)-1,1′-bi-2-naphthol (SBN) binding to planar-supported lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphotidylcholine (POPC) based on the intrinsic chirality of the molecules. C-SHG adsorption isotherms of RBN and SBN reveal Langmuir adsorption behavior with binding constants of 2.7 ± 0.2 × 10⁵ M⁻¹ and 3.0 ± 0.1 × 10⁵ M⁻¹, respectively. The kinetics of RBN binding to a POPC bilayer was also measured. It was determined that the adsorption rate for RBN was 5.7 ± 0.4 × 10³ s⁻¹M⁻¹ and the desorption rate was 2.1 ± 0.8 × 10⁻² s⁻¹. From the kinetic data a binding constant of 2.7 ± 1.0 × 10⁵ M⁻¹ was calculated, which agrees well with the thermodynamic measurement. The C-SHG technique was correlated with surface tension measurements in order to determine the RBN surface excess within the POPC membrane. The maximum surface excess of RBN in a monolayer of POPC was 4.3 ± 0.5 × 10⁻¹¹ mol cm². Using the maximum surface excess in conjunction with the C-SHG binding data a lower limit of detection of 1.5 ±0.1 × 10⁻¹³ mols cm⁻² was calculated. The results of these studies show that C-SHG is a powerful tool for the study of chiral molecular interactions at surfaces.

A splitless thermal desorber unit that interfaces a differential mobility spectrometry (DMS) sensor has been devised. This device was characterized by the detection of benzene, toluene, and xylene (BTX) in water. The detection of BTX in water is important for environmental monitoring, and ion mobility measurements are traditionally difficult for hydrocarbons in water because water competes for charge and quenches the hydrocarbon signals. This paper reports the use of a DMS with a photoionization source that is directly coupled to a solid-phase microextraction (SPME) desorber. The separation and detection capabilities of the DMS were demonstrated using BTX components. Detection limits for benzene, toluene, and m-xylene were 75, 50, and 5 μg mL⁻¹, respectively.

This paper presents technical developments for the detection of formaldehyde (CH₂O) using laser-induced fluorescence. The easily accessible third harmonic of the Nd:YAG laser at 355 nm was used for excitation of formaldehyde. In order to investigate potential background fluorescence, e.g., from large molecules such as polyaromatic hydrocarbons, special attention was paid to investigating the possibility of scanning the wavelength of a single-mode Nd:YAG laser under the gain profile, ∼3 cm⁻¹, on and off resonance. Furthermore, a technique for simultaneous detection of formaldehyde and OH using one laser system is presented. The single-mode Nd: YAG laser at 355 nm in combination with an optical parametric oscillator (OPO) laser tuned to 283 nm was used for simultaneous two-dimensional imaging of both species using one charge-coupled device (CCD) detector equipped with a dual filter image separator. The techniques are demonstrated with measurements in laboratory flames and the combined measurements are also demonstrated in an engine.

We have designed and demonstrated a standoff Raman system for detecting high explosive materials at distances up to 50 meters in ambient light conditions. In the system, light is collected using an 8-in. Schmidt–Cassegrain telescope fiber-coupled to an f/1.8 spectrograph with a gated intensified charge-coupled device (ICCD) detector. A frequency-doubled Nd: YAG (532 nm) pulsed (10 Hz) laser is used as the excitation source for measuring remote spectra of samples containing up to 8% explosive materials. The explosives RDX, TNT, and PETN as well as nitrate- and chlorate-containing materials were used to evaluate the performance of the system with samples placed at distances of 27 and 50 meters. Laser power studies were performed to determine the effects of laser heating and photodegradation on the samples. Raman signal levels were found to increase linearly with increasing laser energy up to ∼3 × 10⁶ W/cm² for all samples except TNT, which showed some evidence of photo- or thermal degradation at higher laser power densities. Detector gate width studies showed that Raman spectra could be acquired in high levels of ambient light using a 10 microsecond gate width.

The performance characteristics of a kilohertz solid-state laser source for ultraviolet Raman spectroscopy are described. Deep ultraviolet (UV) excitation in the 193–210 nm region is provided by mixing of the fundamental and third harmonics of a Ti–sapphire laser, which is pumped by the second harmonic of a Q-Switched Nd–YLF laser. The combination of tunability, narrow linewidth, high average power, good stability, and kilohertz repetition rate makes this laser suitable for deep UV resonance Raman applications. The short pulse duration (∼20 ns) permits nanosecond time resolution in pump–probe applications. The low peak power and high data rate provide artifact-free spectra with a high signal-to-noise ratio. UV Raman cross-section and Raman excitation profiles are reported for gaseous O₂ (relative to N₂), aqueous ClO₄⁻, tyrosine, phenylalanine, tryptophan, histidine, and hemoglobin excited between 193 nm and 210 nm to illustrate laser performance.

Surface-enhanced Raman scattering (SERS) has been studied using a silver-coated porous glass-ceramic material as a new type of substrate. The porous glass-ceramic is in the CaO–TiO₂–P₂O₅ system prepared by controlled crystallization and subsequent chemical leaching of the dense glass-ceramic, leaving a solid skeleton with pores ranging in size from 50 nm to submicrometer. Silver was coated on the surface of the porous glass-ceramic by radio frequency (RF) sputtering or e-beam evaporation in vacuum. SERS spectra of excellent quality were obtained from several dyes and carboxylic acid molecules, including rhodamine 6G, crystal violet, isonicotinic acid, and benzoic acid, using this new substrate. This new substrate showed a good compatibility with these molecules. The porous glass-ceramic with a nanometer-structured surface accommodated both test molecules and silver film. The absorbed molecules were therefore better interfaced with silver for surface-enhanced Raman scattering.

A number of definitions of multivariate selectivity have been proposed in the literature. Arguably, the one that enjoys the greatest chemometric attention has been the net analyte signal (NAS) based definitions of Lorber and Zinn. Recent works have suggested that similar inference can be made for inverse least-squares calibration methods (e.g., principal components regression). However, the properties of inverse calibration methods are markedly different than classical methods, so in many practical cases involving inverse models classically derived figures of merit cannot be transparently interpreted. In Part I of this work, we discuss a selectivity framework that is theoretically consistent regardless of the calibration method. Importantly, it is also experimentally measurable, either through controlled selectivity experiments, or through analysis on opportunistically acquired sample measurements. It is statistically advantageous to use the former if such control is achievable. Selectivity is defined to be a function of the change in predicted analyte concentration that will result from a change in the concentration of an interferant, an approach consistent with traditional definitions of analytical selectivity and National Committee for Clinical Laboratory Standards recommendations for interference testing. Unlike the NAS-based definition of selectivity, the definition discussed herein is relevant to only a particular analyte–interferant pair. The theoretical and experimental aspects of this approach are illustrated with simulated data herein and in Part II of this paper, which investigates several experimental near-infrared data sets.

In Part I of this paper, a framework for multivariate selectivity was introduced that is both calculable from first principles and experimentally tractable. In this part, we employ the proposed selectivity framework for analyzing both in vitro and in vivo near-infrared experimental data. Two in vitro data sets are used to compare different methods for estimating selectivity and to demonstrate the benefits obtained from validation data with expanded interferant concentration ranges. The in vitro data also demonstrate that the experimentally estimated selectivities provide insights into the properties of the calibration models that are difficult or impossible to infer by other means. The merits of the proposed selectivity function are further demonstrated using a complex in vivo application: the noninvasive measurement of ethanol in humans. Results indicate that in vivo calibration model sensitivity, selectivity, and concentration correlations can be systematically interrogated using the proposed selectivity framework and judicious use of experimental measurements. These analyses not only provide selectivity and sensitivity information, but also the variance components of the total MSEP, which is invaluable information for both method development and analytical method characterization.

The traditional way of handling temperature shifts and other perturbations in calibration situations is to incorporate the non-relevant spectral variation in the calibration set by measuring the samples at various conditions. The present paper proposes two low-cost approaches based on simulation and prior knowledge about the perturbations, and these are compared to traditional methods. The first approach is based on augmentation of the calibration matrix through adding simulated noise on the spectra. The second approach is a correction method that removes the non-relevant variation from new spectra. Neither method demands exact knowledge of the perturbation levels. Using the augmentation method it was found that a few, in this case four, selected samples run under different conditions gave approximately the same robustness as running all the calibration samples under different conditions. For the carbohydrate data set, all robustification methods investigated worked well, including the use of pure water spectra for temperature compensation. For the more complex meat data set, only the augmentation method gave comparable results to the full global model.

Visible and near-infrared (NIR) integrating sphere spectroscopy and chemometric multivariate linear regression were applied to determine hematocrit (HCT) and oxygen saturation (SatO₂) of circulating human blood. Diffuse transmission, total transmission, and diffuse reflectance were measured and the partial least squares method (PLS) was used for calibration considering different wavelength ranges and selected optical measurement parameters. HCT and SatO₂ were changed independently. Each parameter was adjusted to different levels and four designs with blood from different donors were carried out for the calibration with PLS. The calibration included the changes in hemolysis as well as inter-individual differences in cell dimensions and hemoglobin content. At a sample thickness of 0.1 mm the HCT and SatO₂ were predicted with a root mean square error (PRMSE) of 1.4% and 2.5%, respectively, using transmission and reflectance spectra and the full Vis-NIR range. Using only diffuse NIR reflectance spectroscopy and a sample thickness of 1 mm, HCT and SatO₂ could be predicted with a PRMSE of 1.9% and 2.8%, respectively. Prediction of hemolysis was also possible for one blood sample with a PRMSE of 0.8% and keeping HCT and SatO₂ stable with a PRMSE of 0.03%.

Near-infrared reflectance spectroscopy was evaluated for its effectiveness at predicting pre-visual decline in eastern hemlock trees. An ASD FieldSpec Pro FR field spectroradiometer measuring 2100 contiguous 1-nm-wide channels from 350 nm to 2500 nm was used to collect spectra from fresh hemlock foliage. Full spectrum partial least squares (PLS) regression equations and reduced stepwise linear regression equations were compared. The best decline predictive model was a 6-term linear regression equation (R² = 0.71, RMSE = 0.591) based on: Carter Miller Stress Index (R694/R760), Derivative Chlorophyll Index (FD705/FD723), Normalized Difference Vegetation Index ((R800 – R680)/(R800 + R680)), R950, R1922, and FD1388. Accuracy assessment showed that this equation predicted an 11-class decline rating with a 1-class tolerance accuracy of 96% and differentiated healthy trees from those in very early decline with 72% accuracy. These results indicate that narrow-band sensors could be developed to detect very early stages of hemlock decline, before visual symptoms are apparent. This capability would enable land managers to identify early hemlock woolly adelgid infestations and monitor forest health over large areas of the landscape.