Transflection sampling

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. 

Solid samples can also be measured in transmission, although reflection or transflection measurements are more common. Open arrangements with the source on one side of the sample and the spectral analyser on the other side are prevalently used, e.g. in industrial process control. For absolute quantitative analysis the thickness of the object must either be constant or be measured. 

For some applications, especially such involving solid samples or fluids containing suspended particles, reflection spectroscopic systems are better suitable than transmission sensors. Apart from specular reflection, which provides comparatively little information and is of hardly any practical importance for IR sensing, two reflectrometric methods can be used to gain spectroscopic information about a sample diffuse reflection and transflection, a combination of transmission and diffuse reflection. 

Transflection is essentially a cross between transmission and mirror reflection. When light is shined onto a reflective surface covered by an optically clear sample, either in liquid or in solid form, two processes occur -... 

In practice, very few applications of FEWS sensors can be found outside laboratory applications and demonstration systems. In the near-IR, suitable fibres are readily available but usually there is no real necessity to use them. Possible transmission pathlengths are sufficiently large to allow using standard transmission probes, while turbid samples can be measured using transflection or diffuse reflection probes. In the mid-IR, high intrinsic losses, difficulties in fibres handling and limited chemical and mechanical stability limit the applicability of optical fibres as sensor elements. 

Like the filtered flow cell, the filtered immersion probe (either transflection or transmission) may be used in sample environments in which bubbles or particles make the use of unfiltered samples impossible. 

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. 

The samples used to construct the models should be similar to the production samples also, their spectra should be recorded in the same mode (reflectance, transmission or transflectance) as those for the samples to be subsequently predicted, and include all potential sources of variability. Although such sources are relatively limited - pharmaceutical samples usually have a well-defined qualitative and quantitative composition from raw material to end product, and production processes are solidly established and reproducible - their... 

Semisolid samples. As with liquid samples, methods (B) and (C) are the best choices for this type of sample. The specific choice will depend on fhe rheological properties (viscosity, density, air retention) of the particular preparation. These samples are best measured in the transflectance mode. Liquid and semisolid samples may contain a mixture of solvents of disparate volatility which may evaporate separately during the measurement process. Differences in solvent volatility can alter the sample matrix and lead to errors in the determination which are best avoided by using a set of calibration samples spanning an expanded range of solvent proportions. ... 

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... 

Beyers et aV° in the Polymer Research Division of BASF-AG used in-line transflectance NIR to monitor methyl methacrylate (MMA) and iV,7V-dimethylacrylamide (DMAAm) monomers in a copolymerization reaction. The work in this paper is of interest as it illustrates an example of calibration development done off-line with a very limited number of prepared calibration samples. The value of the measurement is to control the end properties of the products resulting from the copolymerization reaction. The end properties are related to many parameters including the intramolecular chemical composition distribution (CCD). 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. 

Transflection is essentially a cross between transmission and reflection. When light is shined onto a reflective surface covered by an optically clear sample, two processes occur - reflection off the top surface of the sample and transmission and reflection off the mirror, followed by a second transmission. As for most materials the surface reflection is low in comparison to the reflection off the mirror, the total collected radiation corresponds to a transmission measurement with double pathlength. Instead of a transparent support, transflection systems require only a spectrally neutral, or at least constant, broadband reflector under the sample. 

Fortunately, automated fiber-optic probe-based dissolution systems have begun to appear for these solid dosage-form applications. One such system uses dip-type UV transflectance fiber-optic probes, each coupled to a miniature photodiode array (PDA) spectrophotometer to measure drug release in real time. This fiber-optic dissolution system can analyze immediate- and controlled-release formulations. The system is more accurate and precise than conventional dissolution test systems, and it is easier to set up than conventional manual sampling or automated sipper-sampling systems with analysis by spectrophotometry or HPLC. 

The final type of measurement that can be made with the microscope in its reflection mode is diffuse reflection (DR) spectroscopy. Today, very few appHca-tions of mid-lR microspectroscopy of neat samples are available, because for mid-IR DR spectrometry the samples should be diluted to a concentration of between 0.5 and 5% with a nonabsorbing diluent (e.g., KBr powder) to preclude band saturation and severe distortion by reflection from the front surface of the particles. However, this mode has substantial application for NIR measurements, where sample dilution is not needed. Because the absorption of NIR radiation by most samples is rather weak, they must either be at least 1 mm thick or be mounted on a reflective or diffusing substrate, such as a ceramic or Teflon disk. In the latter case, the spectrum is caused by a combination of diffuse reflection, transflection and front-surface reflection (hopefully with diffuse reflection being the dominant process). 

Both types are used commonly in the pharmaceutical industry. In an at-line assay, samples are taken and analysed either by transmission/transflectance NIR if liquids, or reflectance if solids. The spectra generated are then used to build identification libraries. These hbraries must include the normal variation found in a good raw material distribution. Using these libraries, correlation or conformity... 

One hundred and thirty-eight (138) oil samples were analyzed with visible (vis) and near-infrared (NIR) transflectance spectroscopy. Forty-six of them were Greek pure extra virgin olive oils and the same oils adulterated with 1% (w/w) and 5% (w/w) sunflower oil. However, no significant difference was found between the spectrum of pure sunflower oil and that of olive oil, which can be detected by the naked eye. Olive and sunflower oils differ in their composition principally in their content of linoleic and oleic acids. Accordingly, typical figures for olive oil were quoted at 12.3% and 66.3%, respectively, while for sunflower oil the corresponding mean values of 66.2% and 25.1%, respectively. 

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). 

A variety of sample presentation methods are available to the analyst. These include transmission (straight and diffuse), reflectance (specular and diffuse), transflectance (reflectance and transmission), and interactance (a combination of reflectance and transmission). These methods are discussed in greater detail in Chapter 7. 

The external reflectance method provides a mirror-style reflection from the surface of the sample. It is used only for samples that have flat, reflective surfaces. The most common applications of external reflectance are for the direct measurement for films, coatings, or surface contaminants on metal surfaces. These measurements are sometimes called transflectance or reflection/absorption (Fig. 4). 

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. 

The NIR researcher, user, and engineer must give attention to sample presentation devices that directly govern the quality of the spectra themselves. The type of sample presentations are mainly divided into transmission, reflection, transflection, and interaction modes as shown in Figure 5.4. 

The transflection mode is the combination of transmission and reflection, which was originally developed by Technicon from the InfraAlyzer. Incident light is transmitted through the sample and then scattered back from a reflector, which is made of ceramic or aluminium to be compatible with the diffuse reflection characteristics of the instrument. The NIR spectra of not only liquid but also turbid material can be detected by the integrating sphere or the detector closely settled to the sample. NIR spectra from small volumes of the sample can be clearly measured. 

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