Combination absorbance-reflectance

Optical transduction modes applied in combination with enzyme based fiber-optic sensors include absorbance, reflectance, fluorescence,... 

More complicated absorptions, with two or more strong absorptions by a single molecule, lead to a more complex interpretation of reflected color, but the concept is the same. A substance will appear to our eyes as the combination of reflected (not absorbed) wavelengths. 

Absorption by molecules removes the absorbed wavelengths from white light, and a sample will appear to our eyes as the combination of reflected wavelengths. 

The last three detection schemes apply only under very special circumstances. Transmission EXAFS is strictly a probe of bulk structure, i.e., more than about a thousand monolayers. The electron- and ion-yield detection methods, which are used in reflection rather than transmission schemes, provide surface sensitivity, 1-1,000 A, and are inherendy insensitive to bulk structure. X-ray fluorescence EXAFS has the widest range of sensitivity—from monolayer to bulk levels. The combination of electron or ion yield and transmission EXAFS measurements can provide structural information about the X-ray absorbing element at the surface and in the bulk, respectively, of a sample. 

From this it follows that the plot of the logarithm of the absorbance change against time gives k, not k2. The rationale is that the analytical method really monitors the entire conversion. The rate constant that characterizes the buildup of P2 reflects the loss of A by all the reactions that consume it. The time at which a given fraction of A has reacted is the same time at which the same fraction of the final P2 has formed. Of course, one can obtain the value of k2 by combining the data for the kinetics and the yield ... 

It is important to stress that ATR absorbance is strongly affected by the sample/crystal contact. Quantitative results are thus difficult to obtain even if the contact is maintained during the sample rotation that is required to record all four polarized spectra. A reference band that does not show significant dichroism is thus most often used to normalize the polarized absorbances in order to obtain quantitative data. For instance, the 1,410 cm-1 band of PET has often been chosen for that purpose, not only for ATR studies but also for specular reflectance (see below) and even transmission studies when the sample thickness is not uniform. It was shown that an appropriate normalization is possible even if no such reference band is available, by using a combination of two bands with orthogonal dichroism [34]. When performing ATR experiments, one should also make certain that the applied pressure does not create artifacts by affecting the structure of the sample. 

As for all absorbance-based spectral measurements, the intensity data represented in a raw (single-beam) chemical image are a combination of the spectral response of both the instrument and the sample. In order to remove the instrument response component, it is necessary to ratio the data to a background reference. For reflectance measurements, the background is a separate data cube typically acquired from a uniform. 

Absorbance signals seen in NIR consist of combination and overtone bands of hydrogen bonds such as C-H, N-H, 0-H, and S-H, which are aroused by large force constants and small mass. NIR spectra thus cover precious information on chemical as well as physical properties of analyzed samples due to characteristic reflectance and absorbance patterns [121-123], which makes this analysis method applicable to the characterization of monolithic stationary phases. 

In diffuse reflection spectroscopy, the spectrometer beam is reflected from, scattered by, or transmitted through the sample, whereas the diffusely scattered light is reflected back and directed to the detector. The other part of the electromagnetic radiation is absorbed or scattered by the sample [124,125]. Changes in band shapes or intensity as well as signal shifts can be affected by morphological and physicochemical properties of the sample or combinations thereof (e.g., chemical absorptions, particle size, refractive index, surface area, crystallinity, porosity, pore size, hardness, and packing density [126]). Therefore, NIR diffuse reflection spectra can be interpreted in dependence of various physical parameters [127]. 

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