Transflection Sampling Technique

The methods of presenting samples such as a tissue or isolated single cell for study in an FT-IR microscope have to date been predominantly confined to transmission and, the so-called, transflection sampling techniques. The latter is actually a reflection-absorption technique vide infra). Of increasing recent interest is use of the so-called ATR sampling technique for the analysis of tissue samples. ATR is an abbreviation for attenuated total reflection and is an internal reflection spectroscopy technique. On the horizon are perhaps nearfield techniques. Each of these will now be considered in turn. 

Figure 2.11 Schematic of the transflection sampling technique. The dashed arrow shows the weak specular reflection component see text for details.

Nonetheless, near-IR is the most widely used IR technique. Less intense water absorptions permit to increase the sampling volume to compensate, to some extent, for the lower near-IR absorption coefficients and the inferior specificity of the absorption bands can for many applications be overcome by application of advanced chemometric methods. Miniaturised light sources, various sensor probes, in particular based on transmission or transflectance layouts, and detectors for this spectral range are available at competitive prices, as are (telecommunications) glass or quartz fibres. 

The procedures used to record NIR spectra for samples are much less labor-intensive than those involved in other spectroscopic analytical techniques. NIR spectral information can be obtained from transmission, reflectance and transflectance measurements (Figure 14.1) this allows the measurement process to be adapted to the physical characteristics of the sample and expedites analyses by avoiding the need for sample preparation. 

NIR spectroscopy is probably the most successful technique for the development of qualitative and quantitative methods in the pharmaceutical industry. NIR spectra contain both chemical and physical information from samples (solid and liquid). Spectra can be acquired off-line in three different modes transmittance, reflectance and transflectance. In all cases, the spectra are obtained in a few seconds without or minimum sample pretreatment. Multivariate data analysis techniques are usually needed for the development of the... 

As the reflected radiation is emitted from the sample in a random direction, diffusely reflected radiation can be separated from, potentially sensor-blinding, specular reflections. Common techniques are off-angle positioning of the sensor with respect to the position(s) of the illumination source(s) and the use of polarisation filters. Application restrictions apply to optically clear samples with little to no scattering centres, thin samples on an absorbing background and dark samples. In either of these cases, the intensity of radiation diffusely reflected off such samples is frequently insufficient for spectral analysis. While dark objectives remain a problem, thin and/or transparent samples can be measured in transmission or in transflectance. 

Specular reflectance techniques basically involve a mirror-like reflection from the sample surface that occurs when the reflection angle equals the angle of incident radiation. It is used for samples that are reflective (smooth surface) or attached to a reflective backing. Thus, specular techniques provide a reflectance measurement for reflective materials, and a reflection-absorption (transflectance) measurement for the surface films deposited on, or pressed against reflective surfaces (Figure 9). 

Needless to say, repetition or reproducibility is a fundamental requirement for sample preparation for NIR measurement. Shenk et al. explained the devices currently available from the various instrument manufacturers for sample presentations (8). These variations of packing techniques may be applicable in several approaches to the sample quality analysis problem. The measurement of diffuse reflectance spectra of dried and ground samples in a closed-cup device is the most commonly used procedure. On the other hand, transflectance and transmission techniques are available to analyze wet or liquid samples. Details of sample presentation devices are described below in this chapter. 

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