Diffuse-reflection measurements principles

A solvent free, fast and environmentally friendly near infrared-based methodology was developed for the determination and quality control of 11 pesticides in commercially available formulations. This methodology was based on the direct measurement of the diffuse reflectance spectra of solid samples inside glass vials and a multivariate calibration model to determine the active principle concentration in agrochemicals. The proposed PLS model was made using 11 known commercial and 22 doped samples (11 under and 11 over dosed) for calibration and 22 different formulations as the validation set. For Buprofezin, Chlorsulfuron, Cyromazine, Daminozide, Diuron and Iprodione determination, the information in the spectral range between 1618 and 2630 nm of the reflectance spectra was employed. On the other hand, for Bensulfuron, Fenoxycarb, Metalaxyl, Procymidone and Tricyclazole determination, the first order derivative spectra in the range between 1618 and 2630 nm was used. In both cases, a linear remove correction was applied. Mean accuracy errors between 0.5 and 3.1% were obtained for the validation set. 

Intensified metabolic control, especially in case of diabetes, demands minimal-invasive or non-invasive methods of analytical measurement. For this goal, a method has been developed to measure the blood glucose content in vivo, in direct contact with the skin, by means of diffuse reflection near infrared (NIR) spectroscopy on the basis of multivariate calibration and neural networks (Muller et al. [1997] Fischbacher et al. [1997] Danzer et al. [1998]). Because no patients with any standard blood glucose value are available in principle, a method of indirect calibration has... 

Summarizing, infrared spectroscopy measures, in principle, force constants of chemical bonds. It is a powerful tool in the identification of adsorbed species and their bonding mode. Infrared spectroscopy is an in situ technique, which is applicable in transmission or diffuse reflection mode on real catalysts, and in reflection-absorption mode on single crystal surfaces. Sum frequency generation is a speciality... 

The principle problem with diffuse reflectance is that the specular component of the reflected radiation, that which does not penetrate the sample, is measured along with the diffuse reflected light which penetrates the sample. Generally, the change in specular reflection with frequency is small except in regions of strong absorption bands where the anomalous dispersion leads to Reststrahlen bands in the specular reflection spectrum. When the Reststrahlen bands are observed, the absorption bands can appear inverted at their center. This effect makes quantitative measurements on samples with strong absorptivity very difficult. 

The principle of the measurement by diffuse reflection of the true absorption spectrum of a finely divided colored solid substance has been worked out by Kortiim (24). The substance is introduced under high dilution (mole ratio = 10 to 10 ) into a very disperse powder (average particle diameter 0.1 p) of a neutral white material, which should scatter unselectively and not absorb light in the spectral region concerned. The true shape of the adsorption spectrum is deduced from... 

Figure 15. Diffuse reflectance distributions used to measure tissue absorption and scattering properties non-invasively. (a,b) principle of the technique, showing light entering a point on the tissue surface and the measured radial distribution of the diffusely reflected (backscattered) light that depends on the tissue absorption and scattering properties, (c) external surface probe (courtesy Dr M. Patterson, Hamilton, Canada), (d) endoscopic probe (courtesy Dr R. Bays and colleagues, Lausanne, Switzerland) in this case the distribution is measured along the probe from light input at the end, with the probe placed flat on the tissue (e.g. esophagus) surface.

Figure 17. The effects of tissue attenuation on fluorescence measurements, (a) graph showing measured fluorescence versus Photofrin concentration in different tissues (after Panjehpour et al., 1993 [12]), (b-d) correction for attenuation by the ratio of fluorescence to diffuse reflectance at two different radial distances (b) principle, (c) prototype instrument, (d) photosensitizer concentration measured in vivo versus known concentration by assay on tissues ex vivo for three different tissue types (after Weersink et al., 1997 [13]).

Principles and Characteristics Diffuse reflectance spectroscopy (DRS) is concerned with the efficient collection of diffusively scattered light, the direction of which is unrelated to that of the incident radiation. The technique of DRS enables IR measurements to be made on diffusively scattering solids such as powdered samples without the need for extensive sample preparation. This weak diffuse radiation is collected in a manner... 

Principles and Characteristics Absorption spectroscopy of both vapour and liquid samples by wavelengths in the UV/VIS range, causing electronic transitions in the sample, can be used to quantify components in a mixture. Optical transmission measurements are preferred to diffuse reflectance, they provide higher sensitivity, more precision and enable monofilament fibre optics to be used. Spectroscopic (UVA IS/NIR) analysis of pellets is more complicated. 

IR spectra of TLC-IR solutes obtained by DR-IR and PA-IR exhibit band intensity distortions in spectral regions in which stationary and mobile phases absorb and may not resemble those obtained for the same substances by KBr pellet transmittance measurements. To avoid stationary and mobile phase interferences, solute transfer methods can be used to physically move separated mixture components from the TLC plate to an infrared transparent substrate. Solute transfer is accomplished by subjecting the developed TLC plate to a second solvent that is applied orthogonally to the direction of the chromatographic mobile phase flow. This causes separated mixture components to move to the edge of the TLC plate where they are collected on IR transparent KCI powder. Figure 3 illustrates the basic principle. Solute spectra subsequently obtained by diffuse reflectance IR analysis are similar to those obtained by KBr pellet transmittance measurements. 

When a flexible chain is attached to a fluorescent dye, the rotational diffusion of the dye is slowed down, but as the chain is extended, approaches an asymptotic limit, since the whole of the chain does not participate in the motion of the dye (6, 7). The extent to which the rotational diffusion of the dye is impeded is then a measure of the rigidity of the chain. If the middle of the chain is tagged with a fluorescent label, the rotational relaxation will reflect, in principle,both the local conformational transition and the rotation of the molecule as a whole. Withp. and p characterizing these two processes, the observed relaxation time will be (8)... 

However, the available information is not limited to translational diffusion coefficients and concentrations. By FCS, a wide range of processes can be studied, spanning a time range from nanoseconds to seconds. In principle, any process at equilibrium conditions, which reflects itself in terms of a change of the detected fluorescence F t), can be measured. Three of the most widely applied FCS-based approaches to extract information from molecules undergoing or participating in d3mamic processes or reactions are outlined in Fig. 8.1. 

Fig. 8.3. FCS Proton exchange kinetics measurements at biological membranes, (a) Principal design of experiment. Liposomes were labeled with one FITC fluo-rophore undergoing fluorescence fluctuations due to protonation/deprotonation. (b) Collection of FCS curves of the vesicles at different pH. The FCS curves reflect singlet-triplet transitions in the microsecond time range, protonation kinetics in the 10-100 ps time range and translational diffusion in the milliseconds time range. Inset measured protonation relaxation rates vs. proton concentration, (c) Principle of the proton collecting antenna effect...

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