Applied Spectroscopy

The Zn-to-Cu ratio in brass was measured by laser ablation inductively coupled plasma atomic emission spectroscopy. The influence of laser beam properties (pulse width, wavelength, and power density) on fractional laser ablation was investigated. The behavior of the Zn/Cu ratio vs. laser power density shows that there are different mechanisms influencing ps and ns laser ablation. With the use of a 30 ns pulse duration from an excimer laser, thermal vaporization appears to be the dominant process in the low-power density region. The Zn/Cu ratio approaches stoichiometry at higher power density, but the ablated mass still remains Zn rich. With a 35 ps pulse Nd:YAG laser, a nonthermal mechanism appears to govern the laser ablation process. When a 3 ns Nd:YAG laser is used, both thermal and nonthermal processes exist. For both 3 ns and 30 ps Nd:YAG lasers, stoichiometric ablation can be achieved at higher power densities.

A possibility for modulation of molecular flow through a capillary by its periodic heating and cooling has been investigated. The Knudsen gas flow (when the length of the molecule free path is greater than the diameter of the capillary) at variable temperatures of the capillary and the linear isotherm of adsorption have been analyzed in terms of diffusion approximation. The analytical expression for the outlet flow has been obtained for any given time dependence of the capillary temperature and inlet flow. It has been demonstrated that such modulation can increase both sensitivity and selectivity of mass spectrometric measurements and could be used for on-line analysis of gas streams.

Poor detection limits of two-photon excited fluorescence in cylindrical capillaries are attributed to photothermal expansion and beam astigmatism. Photothermal expansion is demonstrated for excitation in a 1 cm cell and is inferred for the larger diameter capillaries. Astigmatism is caused by focal differences between rays in a plane longitudinal to the capillary and rays in a plane transverse to the capillary. Data were obtained by integrating the fluorescence from variously sized cylindrical and square capillaries, and by photographing the fluorescence within a 1 cm cell.

Fluorescence anisotropy and intensity decay experiments on proteins can provide detailed information on biomolecule dynamics and function. However, experiments of this sort are normally performed while the biomolecule is at or near equilibrium. Although information on protein dynamics under equilibrium conditions is extremely important, details about the protein behavior while it is actually undergoing change can provide significantly more insight into the overall protein behavior. Multiharmonic Fourier frequency-domain fluorescence provides a means to acquire fluorescence anisotropy and intensity decay information on a reasonably rapid time scale. As a result, one can potentially track protein nanosecond and subnanosecond dynamical processes on-the-fly as they undergo change(s) during, for example, protein–ligand binding, enzymatic reactions, or antigen/hapten–antibody association. To illustrate the potential of the frequency-domain on-the-fly methodology, we report here on the behavior of a model protein, bovine serum albumin, that has been labeled site-selectively with the fluorescent probe acrylodan (BSA-Ac). Conformational changes in the BSA-Ac are effected by using trypsin or β-mercaptoethanol (BME). BME is a disulfide interchange reagent, and trypsin cleaves and excises from the entire BSA molecule a 21 amino acid peptide segment that contains the covalently attached Ac residue. This paper focuses on the time course of the fluorescence anisotropy and intensity decay kinetics of BSA-Ac as it reacts with trypsin or BME.

Recent clinical trials have demonstrated the potential of fluorescence spectroscopy for in vivo diagnosis of pathology. There is significant potential to reduce the cost and complexity of instrumentation to measure tissue spectra; however, careful analysis is required to maximize performance and minimize cost. One measure of performance is the signal-to-noise ratio (SNR) of the resulting data. This paper describes a method to predict the SNR of a given optical design for a particular tissue application. In order to calculate the expected SNR, two pieces of information are required: (1) the throughput and inherent noise of the system and (2) a quantitative relationship between the illumination energy and the resulting tissue fluorescence available for collection, which we define as the tissue fluorescence efficiency (FE). We present a method to calculate the fluorescence efficiency of tissue from in vivo measurements of tissue fluorescence. We report FE measurements of the normal and precancerous human cervix in vivo at 337, 380, and 460 nm excitation. We also present and evaluate a method to estimate the throughput and noise of various spectrometers and predict the expected SNR for tissue spectra by using the measured tissue FE. For squamous cervical tissue, as the degree of the disease increases, FE decreases, and as the excitation wavelength increases, FE decreases. Cervical tissue FE varies more than two orders of magnitude, depending on the tissue type and on the excitation wavelength used. Our SNR calculations, based on measured values of tissue FE, demonstrate agreement within a factor of 1.3 of the measured SNR on average. This method can be used to estimate the performance of different spectrometer designs for clinical use.

Water-soluble sulfonated calix[4]arene (SCX4) was used as the host molecule for the guest dye(probe), Brilliant Cresyl Blue (BCB), which is commonly employed for studies of various biological systems. Absorption and fluorescence techniques were employed for the analysis of this system. The formation of a complex between the BCB dye and SCX4 results in a decrease in the BCB fluorescence intensity. A study of the changes in absorption and of temperature effects on the fluorescence of BCB/SCX4 was conducted. The binding constant for BCB/SCX4 at room temperature was estimated to be 8.49 × 10³ M⁻¹.

A simple and inexpensive sapphire test cell has been developed for the performance of in situ Raman spectroscopy within aqueous environments up to the conditions of 400 °C and 345 bar pressure. The cell volume is approximately 11 cm³ and is held by a heating enclosure within the optical path of a Raman spectrometer. With this cell, Raman vibrational measurements have been satisfactorily performed on aqueous samples and on solid substrates within aqueous environments. The test cell and its supporting equipment are fully described, as well as the Raman data manipulation method used for the processing of high-temperature aqueous Raman measurements.

The IR spectrum of a sample in acidic and alkaline solutions cannot be retrieved adequately when only the spectrum of pure water is subtracted. After such an operation, some water bands remain in the spectrum, which also has a distorted baseline. An analysis of a series of IR spectra of HCl and NaOH solutions showed that they could be represented by two pairs of eigenspectra, one pair for the acidic solutions and the other for the basic solutions. The fraction of each eigenspectrum of a sample in an acidic or alkaline solution is determined with the 2100 and 3300 cm⁻¹ water bands. After subtraction, no baseline adjustment is necessary. The effectiveness of the method used to subtract the water bands is illustrated with solutions of malic acid at low and high pH.

A baseline correction tool using the method of Bézier has been developed to permit the drawing of curves in a manner that allows full control of the line curvatures by the user. The basic concepts and principle of the Bézier algorithm are explained as well as the modifications that were made to take into account the limitations imposed by the treatment of spectral data. The application of this particular curve drawing routine to an existing baseline correction module offers facilities and extended capabilities that typical polynomial methods using least-squares fitted lines cannot attain. The examples of applications that are given demonstrate how this method can be performed, as well as its efficiency.

Step-scan transient Fourier transform infrared (FT-IR) difference spectra are often measured in an ac-coupled configuration. The resulting differential intensity spectra contain both positive and negative bands. This condition poses problems for direct phase correction by the standard Mertz and Forman methods. Restricting the calculated phase angle to the range [–π/2, π/2] was previously shown to fix some of these problems, but we show that the use of a reduced-resolution phase spectrum can produce other artifacts. The effect of reduced resolution is analyzed for a simulated noise-free spectrum and for a measured transient spectrum of a real photochemical system, bacteriorhodopsin. Examination of these results reveals that the Mertz and Mertz Signed methods can produce spectral bands of reduced magnitude and unusual band shape, with considerable amounts of intensity remaining along the imaginary axis after phase correction. However, these errors can be eliminated by self-convolution of the measured interferogram, which doubles all phase angles, prior to smoothing. This procedure removes the potential discontinuities in the phase angle due to sign changes in the differential spectrum. With bacteriorhodopsin, this doubled-angle method for direct phase correction is able to produce a transient spectrum which closely matches that produced by using a separately measured dc interferogram to calculate the phase angle.

Fourier transform infrared spectroscopy (FT-IR) was used to study the cure reaction process of an ester-type photosensitive polyimide, PMDA/ODA films coated on silicon wafer substrates. In the in situ high-temperature FT-IR study carried out in vacuum, all the characteristic peaks of imide and the crosslinkable group were observed to decrease gradually with an increase in temperature. The completion of the imidization reaction is at ∼270 °C. Our result shows that the ester-type photosensitive polyimide can be cured at a much lower temperature than previously reported. The imide peak δ_as(C, C > NC) at 588 cm⁻¹ was used to follow the imidization process, and the result agrees well with that obtained by using another imide peak v(C–N–C) at 1378 cm⁻¹. The film shrinks by one third during the cure process, and the temperature dependence of the film thickness shows excellent agreement with the “imidization degree”.

Development of varieties of high-erucic-acid (HEAR) rapeseed with high levels of erucic acid (C22:1) would be valuable for use in oleochemical feedstocks. As an aid to this breeding effort, a rapid method to detect trierucin (C22:1 triacylglycerol), an indicator that erucic acid is being incorporated in all three positions of the triacylglycerol, would be useful. Fingerprint (1850–600 cm⁻¹) Fourier transform infrared (FT-IR) spectra of HEAR oil extracted from Brassica napus L. cv. Mercury, trierucin, and varying amounts of trierucin spiked in this oil were recorded. The regions of 1790–1690 cm⁻¹ and 1485–990 cm⁻¹ were subjected to a partial least-squares (PLS) procedure to obtain a calibration equation [R² = 0.992 and root-mean-squared deviation (RMSD) = 2.78]. The established calibration was then tested on the spectra of mixtures of another HEAR oil from B. napus cv. Turret and trierucin. The predicted values differ from the actual values with an RMSD of 1.6%. The calibration was also tested on high-erucic-acid oils from crambe (Crambe abyssinica) and nasturtium (Tropaeolum majus L.) seeds. For crambe, the value found (0.4%) was close to that reported in the literature, while the value of 75% for nasturtium oil was close to the value for total triacylglycerols with erucic acid in the 2-position as reported in the literature. FT-IR spectra of triolein (C18: 1 triacylglycerol), trieicosenoin (C20:1 triacylglycerol), and trinervonin (C24:1 triacylglycerol) compared with the FT-IR spectrum of trierucin revealed the uniqueness of IR spectrum of trierucin that forms the basis of PLS analysis of trierucin in HEAR oils.

The first paper in a series devoted to self-association in neat butanols presents the results of two-dimensional (2D) near-infrared (NIR) correlation analysis of temperature-induced spectral variations of sec-butanol. By taking advantage of resolution enhancement in the 2D correlation spectra, it was possible to identify spectral features due to vibrations of the free and associated OH groups in the first-overtone region. On the basis of a few assumptions, band assignments of the various types of OH bonds have been proposed. The monomer band (near 7100 cm⁻¹) can be resolved into three components; two of them are due to a rotational isomerism (7089 and 7116 cm⁻¹), and the third one is attributed to the free terminal OH groups in linear polymers (7055 cm⁻¹). The presence of the 7055 cm⁻¹ band implies that the intensity of the monomer peak cannot be used as a measure of the concentration of the monomer species (except in very diluted solutions). Thus, previous estimations of equilibrium constants and thermodynamic parameters associated with hydrogen-bond dissociation have been subject to unacceptable error. At higher temperatures, a new band near 6550 cm⁻¹ becomes visible. This band originates from bended OHO bond, mostly in the cyclic polymers. In order to obtain more detailed information on the complex mechanism of the thermal dissociation of hydrogen-bonded sec-butanol in the pure liquid phase, the entire experimental temperature range was divided into narrower ranges, and then 2D correlation analysis was performed for smaller data sets. It has been shown that the variations of population of the polymeric species and the cyclic dimers are faster than the corresponding changes for the monomers. At elevated temperatures an appreciable dissociation of the cyclic species takes place.

Near-infrared (NIR) reflectance spectroscopy has been used to differentiate water in different hydrogen-bonding environments of skin in vivo and to determine the effect of moisturizer application on water content. Four types of water have been observed in the second-derivative spectrum of skin. These are assigned as water associated with the lipid bilayers, primary and secondary water of hydration on protein, and bulk (free) water in deeper tissue. The intensities of the lipid-associated water band and the bulk water band depend on anatomical site. The latter, particularly, may be affected by corneocyte size and may have potential in predicting transdermal penetration of drugs. Moisturizer application appears to have little effect on water content, but results in changes in the degree of scattering of radiation, suggesting that moisturizers smooth, rather than hydrate, the skin.

We propose a high-resolution, wide-bandwidth, high-optical-throughput spectroscopic system, in which a Fabry–Perot interferometer (FPI) is used in tandem with a second spectrometer. As the second spectrometer, one of the following three instruments can be used: (1) a grating dispersion-type monochromator attached to a multichannel detector (MCD), (2) a conventional Fourier transform spectrometer (FTS), or (3) a multichannel FTS (MFTS). The principle of operation for the three systems is the same. First, we adjust the free spectral range (FSR) of the FPI to be k (>1) times the data interval DI (or resolved wavenumber) of the second spectrometer. The FSR is divided by N (>k) equally, and the etalon plate spacing of the FPI is changed N times with a step interval of FSR/N in wavenumber. Thereby we gather a total of N spectra from the second spectrometer. Finally, a single high-resolution spectrum is synthesized from the N spectra. To demonstrate the real capabilities of the proposed systems, we present computer simulation results.

Line patterns with spacings ranging from 10 to 50 μm have been achieved by 488 nm photolysis of porous glasses impregnated with Fe(CO)₅. Photolysis was accomplished with a focused Ar⁺ laser impinging onto a sample mounted on a computer-controlled X-Y stage programmed to create a line pattern. The photodeposited grating was tested by generating a diffraction pattern with a He–Ne laser, which was then used to calculate the grating line spacings. The measured line spacings were found to be in excellent agreement with the spacings defined by the computer-driven stage. Consolidation of the porous glass occurs at 1200 °C, and decreases the sample volume as much as 35%. Line spacing decreases in direct proportion to the decrease in the dimension perpendicular to the grating lines of the sample and consolidation yields gratings that are capable of diffracting light, stable under a variety of conditions, and unaffected by high laser powers.