Transmission microspectroscopy

Raman spectroscopy s sensitivity to the local molecular enviromnent means that it can be correlated to other material properties besides concentration, such as polymorph form, particle size, or polymer crystallinity. This is a powerful advantage, but it can complicate the development and interpretation of calibration models. For example, if a model is built to predict composition, it can appear to fail if the sample particle size distribution does not match what was used in the calibration set. Some models that appear to fail in the field may actually reflect a change in some aspect of the sample that was not sufficiently varied or represented in the calibration set. It is important to identify any differences between laboratory and plant conditions and perform a series of experiments to test the impact of those factors on the spectra and thus the field robustness of any models. This applies not only to physical parameters like flow rate, turbulence, particulates, temperature, crystal size and shape, and pressure, but also to the presence and concentration of minor constituents and expected contaminants. The significance of some of these parameters may be related to the volume of material probed, so factors that are significant in a microspectroscopy mode may not be when using a WAl probe or transmission mode. Regardless, the large calibration data sets required to address these variables can be burdensome. 

FTIR Microspectroscopy.3 A microscope accessory coupled to a liquid-nitrogen-cooled mercury-cadmium-telluride (MCT) detector can be used to obtain an IR spectrum. This is possible in both the transmission and reflectance modes. Several beads are spread on an IR-transparent window (NaCl, KBr, diamond) and possibly flattened via a hand-press or a compression cell. The IR beam is focused on a single bead using the view mode of the microscope. The blank area surrounding the bead is isolated using an adjustable aperture, and a spectrum is recorded using 32 scans (<1 min). A nearby blank area of the same size on the IR transparent window is recorded as the background. 

Schumacher, M., Christl, I., Scheinost, A. C., Jacobsen, C., and Kretzschmar, R. (2005). Chemical heterogeneity of organic soil colloids investigated by scanning transmission X-ray microscopy and C-ls NEXAFS microspectroscopy. Environ. Sci. Technol. 39, 9094-9100. 

Of course, most samples for microspectroscopy are not accessible for transmission measurements, particularly in those circumstances where modification of the sample occurs by preparation of a thin film. 

While IR transmission spectroscopy is a general analytical method for resin samples, internal reflection spectroscopy is especially suited for solid polymer substrates known as pins or crowns. Single-bead analysis is best done by IR microspectroscopy, whereas photoacoustic spectroscopy allows totally nondestructive analysis of resin samples. 

Sample preparation is perhaps the most critical part of a successful IR microspectroscopy experiment In the same way that the IR microscope can be used in a number of ways to collect spectra, so too can sample preparation can approached in a variety of ways. As biological materials are most frequently probed in either transmission mode or reflection mode, these methods will be described here. Other methods are also available, however, such as grazing incidence and attenuated total reflection (ATR) [1]. 

As far as microspectroscopy is concerned, only transmission and reflection under near-normal incidence are initially implemented. The recent launch of specialised objectives provide the user with additional techniques such as external reflection under grazing angle andATR. 

FTIR microspectroscopy (or FTIR microscopy or /r-FTIR) has been a conventional method for materials characterization since 1984, when Analect Instruments (now KVB) introduced a transmission microscope interfaced to its AQS FTIR [181]. Since then, FTIR microspectroscopy has developed into a greatly advanced tool for the analysis of thin films on a wide variety of snbstrates (including a single particle, cell, bacterium, or fiber) for scientific, industrial, and forensic applications [182-I9I]. Examples include oxide layers on technical Si wafers [192], organic films on Si (001) [193], organic [194-196]... 

Figure 4.34. Schematic diagram of on-axis Cassegrain optics with diamond anvil cell (DAC) for IR transmission microspectroscopy Ci and C2 are Cassegrain mirrors. Reprinted, by permission, from J. C. Chervin, B. Canny, J. M. Besson, and Ph. Pruzan, Rev. Sci. Instrum. 66, 2595-2598 (1995), p. 2596, Fig. 1. Copyright American Institute of Physics.

Fraser DJJ, Norton KL, and Griffiths PR (1988) HPLC/FT-IR measurements by transmission, reflection-absorption, and diffuse reflection microscopy. In Messerschmidt RG and Harthcock MA (eds.) Infrared Microspectroscopy Theory and Applications, pp. 197-210. New York Dekker. 

Infrared microspectroscopy has been applied to the study of macerals (organic fractions) from a wide range of coals [106,116] and oxidized coal [117]. It may be performed either in transmission or reflection [106]. In the former case, sample preparation is tedious, and transmission IR microspectroscopy is seldom used. The preparation of samples for reflection measurements is simpler for example, the technique of attenuated total reflectance (ATR) has been applied to the study of coal [106]. Indeed, if the standard ATR accessories (which have been available for many years) were not suitable for block coal samples, recently, an ATR lens, equipped with a silicon (or germanium) internal reflection element, has become available for use with IR microscopes, and the technique was successfully applied... 

Figure 14.33 a) ATR spectra of single nylon carpet fiber untreated (top), treated (middle), and the result of spectral subtraction (bottom), b) Bicomponent fiber transmission spectrum (top), ATR spectrum of Nylon 6 sheath (middle), and difference spectrum of PET core (bottom).Reproduced with permission from Cho, L, et a ., "Single Fiber Analysis by Internal Reflection Infrared Microspectroscopy," iourna of Forensic Sciences 46 (2001), 1309-1314. Copyright 2001, ASTM International. 

The infrared spectrum of each sample was analyzed with a Fourier transform infrared (FTIR) microspectroscopy (IRT-5000-16/FTIR-6200, Jasco Co., Tokyo, Japan) equipvped with a mercury cadmium telluride (MCT) detector via a transmission technique (Gao Lin, 2010 Lin et al., 2006, 2010). All the FTIR spectra were obtained at a 4 cmi resolution and at 100 scans. The components and relative compositions of each sample were estimated quantitatively within the 1740-1600 cm-i region of FTIR spectra by a curve-fitting algorithm with a Gaussian-Lorenzian function (Cheng et al., 2008 Hu et al., 2002). The best curvefitting procedure was performed by iterative fits toward a minimum standard error. The relative composition of the component was computed to be the fractional area of the corresponding peak, divided by the sum of the area for all the peaks. 

The coupling of vibrational spectroscopy with microscopy allows the chemical identification of species in small or microscopically heterogeneous samples. Furthermore, vibrational imaging techniques allow the determination of the spatial distribution of such species, with unique capabilities in terms of chemical contrast and specificity. Molecular microspectroscopy can look at thin transparent samples by transmission or at thick samples using reflection methods, so a wide range of samples can be studied. 

This technique has been used for the identification of undispersed particles in plastic applications. It is particularly useful for resolving customer complaints as well as process development problems. As a general rule particles 20 microns or larger can be analyzed by FT-IR spectroscopy. The approach is to focus the IR beam onto the particle of interest using the microscope, and then scan the FT-IR spectra several times (100 scans), either in transmission or reflectance mode. The sample is then moved slightly to another position and the microscope is focused on a portion of the sample without a defect. The FT-IR spectrum of this part of the sample is recorded in exactly the same way as that of the defective part. The spectrum of the non-defective part is then subtracted from spectrum of the defective part of the sample. The difference spectrum is then used to identify the spot or particle in the defective part. Optical microscopy is often used together with FT-IR microspectroscopy to aid in selecting the area of interest to be analyzed. 

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