Transmission Sampling Technique

The preparation of a tissue specimen of appropriate thickness from such as a biopsy sample (see also Chapter 5) usually follows standard histology practices, particularly since it is a common practice to microtome two parallel adjacent sections of near equivalent thickness. One of these sections is stained in a conventional way, most commonly with haematoxylin and eosin (H E), for 

Glass microscope slides are totally opaque to almost all mid-IR radiation, so the most common substrate on which to support an unstained tissue section for a mid-IR spectroscopic examination is a thin polished window of Cap2 or Bap2 (of typical thickness ca. 0.5 mm). 

Individual cells may also be deposited onto a Cap2 window support, but perhaps the more practical and most appropriate (in terms of its match to cytological practices) method nowadays is to use a low-e glass slide and record a transflection spectrum, see next section. 

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Investigations of the acidity of specific surface sites may be accomplished by studies coordinated with spectroscopic methods, such as infrared (JR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, or mass spectrometry (MS). Surface characterization with Fourier transform infrared (FTIR) spectroscopy can provide quantitative results with experimental methods that are easily performed. However, the transmission sampling techniques traditionally employed for infrared studies may introduce experimental artifacts on the analyzed surface (10,... 

Capillary Thin Film A transmission sampling technique used to obtain spectra of liquids. Typically, a drop of liquid is placed between two infrared transparent windows, which are then placed directly into the infrared beam. The capillary action of the liquid holds the two windows together. 

Cast Films A transmission sampling technique used to analyze polymer films. The polymer is dissolved in a solvent, and the solution is evaporated onto an infrared transparent window giving a polymer film. The window/film combination is then placed directly in the infrared beam. 

KBr Pellet A transmission sampling technique most commonly used on powders and solids. The technique involves grinding the sample and KBr, diluting the sample in the KBr, then pressing the mixture to produce a transparent pellet. The pellet is then placed directly in the infrared beam. 

Mull A transmission sampling technique where the sample is ground then dispersed in an oil or mulling agent. The oil/sample mixture is then sandwiched between two infrared transparent windows and placed in the infrared beam. 

Sealed Liquid Cells A transmission sampling technique used to obtain the spectra of liquids. The cell consists of two infrared transparent windows held a fixed distance apart by a gasket. The cell is filled with liquid then placed in the infrared beam. 

The ATR technique is a commonly used infrared internal reflection sampling technique. It samples only the surface layer in contact with the ATR element the sampling depth probed is typically of the order of 0.3-3 pm [1]. Unless software corrected, compared with a transmission spectrum, the relative intensity of bands within an ATR spectrum increase in intensity with decreasing wavenumber. Several FTIR instrument companies now supply "ATR-correction" software developed to correct for the different relative intensities of bands observed between ATR and transmission spectra, so that ATR spectra can be more easily compared to and searched against transmission spectra. 

All of the usual sampling techniques used in infrared spectroscopy can be used with FT-IR instrumentation. The optics of the sampling chamber of commercial FT-IR instruments are the same as the traditional dispersive instruments so the accessories can be used without modification for the most part. To make full use of the larger aperature of the FT-IR instrument, some accessories should be modified to accomodate the larger beam. The instrumental advantages of FT-IR allow one to use a number of sampling techniques which are not effective using dispersive instrumentation. Transmission, diffuse reflectance and internal reflectance techniques are most often used in the study of epoxy resins. 

There are several major areas of interfacial phenomena to which infrared spectroscopy has been applied that are not treated extensively in this volume. Most of these areas have established bodies of literature of their own. In many of these areas, the replacement of dispersive spectrometers by FT instruments has resulted in continued improvement in sensitivity, and in the interpretation of phenomena at the molecular level. Among these areas are the characterization of polymer surfaces with ATR (127-129) and diffuse reflectance (130) sampling techniques transmission IR studies of the surfaces of powdered samples with adsorbed gases (131-136) alumina(137.138). silica (139). and catalyst (140) surfaces diffuse reflectance studies of organo- modified mineral and glass fiber surfaces (141-143) metal overlayer enhanced ATR (144) and spectroelectrochemistry (145-149). 

The optical absorption arising from the defect transitions is weak because of the low defect densities and in a thin film cannot be measured by optical transmission. The techniques of PDS, CPM and photoemission yield, described in Section 3.3, have sufficient sensitivity. Photocapacitance, which measures the light-induced change in the depletion layer capacitance, is similarly sensitive to weak absorption (Johnson and Biegelsen 1985). PDS measures the heat absorbed in the sample and detects all of the possible optical transitions. At room temperature virtually all the recombination is non-radiative and generates heat by phonon emission. CPM detects photocarriers and so is primarily sensitive to the optical transitions which excite electrons to... 

Transmission spectroscopy (2) is the simplest sampling technique in IR spectroscopy and is used for routine spectral measurements on diverse samples. Resin samples such as polystyrene or TentaGel (3) beads are usually prepared as a potassium bromide disc (pellet). A small amount, usually 1-3 mg, of finely ground solid sample is mixed with approximately 400 mg powdered potassium bromide and then pressed in an evacuated die under high pressure. The resulting discs are transparent and yield good spectra. 

IR spectroscopy is one of the few analytical techniques that can be used for the characterization of solid, liquid, and gas samples. The choice of sampling technique depends upon the goal of the analysis, qualitative identification or quantitative measurement of specific analytes, upon the sample size available, and upon sample composition. Water content of the sample is a major concern, since the most common IR-transparent materials are soluble in water. Samples in different phases must be treated differently. Sampling techniques are available for transmission (absorption) measurements and, since the advent of FTIR, for several types of reflectance (reflection) measurements. The common reflectance measurements are attenuated total reflectance (ATR), diffuse reflectance or diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), and specular reflectance. The term reflection may be used in place of reflectance and may be more accurate specular reflection is actually what occurs in that measurement, for example. However, the term reflectance is widely used in the literature and will be used here. 

These are the oldest and most basic sampling techniques for IR spectroscopy and apply to both FTIR and dispersive IR systems. Transmission analysis can handle a wide range of sample types and can provide both qualitative and quantitative measurements. Transmission analysis provides maximum sensitivity and high sample throughput at relatively low cost. There is in some cases substantial sample preparation required. 

The sample techniques just described are designed for collection of transmission (absorption) spectra. This had been the most common type of IR spectroscopy, but it was limited in its applicahons. There are many types of samples that are not suited to the convenhonal sample cells and techniques just discussed. Thick, opaque solid samples, paints, coahngs, hbers, polymers, aqueous solutions, samples that cannot be... 

Attenuated total reflectance (ATR) accessories are especially useful for obtaining IR spectra of samples that cannot be readily examined by common transmission methods. Such accessories are suitable for studying thick or highly absorbing solid and liquid samples, including films, coatings, powders, threads, adhesives, polymers, and aqueous samples. ATR requires only little sample preparation for most samples, it is one of the most versatile sampling techniques. 

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