Internal reflection experimental

Recently, a formalism has been developed to determine the second and the fourth order parameters of films using polarized total internal reflection fluorescence (TIRF) [71]. Similarly to IR-ATR spectroscopy (Section 4), the experiment makes use of p- and s-polarized excitation, but the fluorescence emission (analyzed either in p- or s-direction) is detected normal to the substrate. Two approaches are developed based on the measurements of two intensity ratios. In the first one, the S angle has to be known experimentally or theoretically, and the order parameters (P2) and (P4) can be determined. In the second one, the order parameter (R ) is obtained by another technique, for instance IR-ATR spectroscopy, which allows deducing the order parameter (P4) and (cos2<5). 

Using internal reflection, also known as attenuated total reflection (ATR), an official ATR-FTIR method (AOCS, 1999a AOAC International, 2000) was recently developed (Mossoba et al., 1996, 2001b Adam et al., 2000) to rapidly (5 min) measure the 966 cm-1 trans band as a symmetric feature on a horizontal baseline (Fig. Dl.7.1 A). The experimental aspects of this ATR infrared official method are far less complex than those involving the conventional transmission measurements. This approach entails (1) ratioing the trans test sam-... 

Protein Adsorption. The development of medical implant polymers has stimulated interest in the use of ATR techniques for monitoring the kinetics of adsorption of proteins involved in thrombogenesis onto polymer surfaces. Such studies employ optical accessories in which an aqueous protein solution (93) or even ex - vivo whole blood (94-%) can be flowed over the surface of the internal reflection element (IRE), which may be coated with a thin layer of the experimental polymer. Modem FT-IR spectrometers are rapid - scanning devices, and hence spectra of the protein layer adsorbed onto the IRE can be computed from a series of inteiferograms recorded continuously in time, yielding ah effective time resolution of as little as 0.8 s early in the kinetic runs. Such capability is important because of the rapid changes in the composition of the adsorbed protein layers which can occur in the first several minutes (97). 

Experimental work has shown that the analysis of quartz in respirable dust by Infrared spectroscopy using a Multiple Internal Reflectance Accessory is a viable technique that is sensitive, accurate and simple to perform. Linearity of a calibration curve from 0 to 200 micrograms has been demonstrated. A detection limit of approximately ten micrograms of quartz was obtained. An accuracy of + 35% at a 95% confidence level was demonstrated by data obtained from participation in the NIOSH PAT Program. 

The experimental aspects of the performance of in situ FTIR measurements are described in Refs. 43 and 44. Figure 13 shows a typical cell for in situ external reflectance mode (e.g., SNIFTIRS type measurements) [94,95], The experimental aspects of its use are described in Ref. 96. Figures 14 and 15 show cells for in situ internal reflectance modes multiple internal reflectance ATR and single internal... 

SHG is a coherent process and in principle the experimental system needed to observe the response is very simple. The fundamental radiation from a laser source incident at an interface generates the harmonic beam via non-linear polarization of the medium. Typically, this beam is observed in reflection, but many studies have been undertaken in total internal reflection and transmission geometries. As the harmonic beam is well separated from the fundamental in frequency, it can be detected the difficulties arise due to the inherent inefficiency of the harmonic generation and the low intensities that need to be detected. The sensitivity and selectivity of SHG to the interfacial species in the presence of the same species in the bulk phase provides the driving force to overcome these experimental difficulties. 

Figure 4.8-11 Experimental arrangement.s for in situ external and internal reflection. spectroscopy. C cup, M metal sheet, PF polymer film, DS doping solution, W window, L IR beam.

With ellipsometry the polarization state of reflected radiation rather than just its intensity, is experimentally determined. Ellipsometry is not so much another experimental technique but a more thorough variety of the traditional ones, whether external or internal reflection. Two results per resolution element, namely the ellipsometric parameters (cf. Eq. 6.4-17) and A, are derived independently from the measurements. These can further be evaluated for the two optical functions of the medium behind the reflecting surface or other two data of a more complex sample. In any case there is no information necessary from other spectral ranges as it is for Kramers-Kronig relations. In comparison to the conventional reflection experiment, ellipsometry grants more information with a more reliable basis, e.g. since no standards are needed. 

Hence, in contrast to transmission spectroscopy, where the sample intercepts the path of the IR beam, in ATR the sample is placed on an IR-transparent crystal (IRE, internal reflection element), permitting total internal reflection. In vivo (or in situ) experimentation is permitted by virtue of one of several design arrangements (a) the region of skin under study may be placed directly in contact with the crystal (b) a remote fiber-optic probe (with IRE head) may be used to transfer the IR beam from the source to the sample and ultimately to the detector and (c) samples removed from the subject may be placed directly onto the crystal. 

A parallel experimental effort to study the internal motion may also demand design ingenuity. To be realistic, the droplets under testing are to be small (of the order of 100 /un), free from any suspension device, and are subjected to the shearing action of high-speed gas stream. Hence observation of stationary droplets may not be easy. Furthermore, direct optical probing of the droplet interior may also be diflBcult because of the severe degree of internal reflection for the small droplets involved. 

The only piece of experimental evidence for the orientation of apoA-I helices on DMPC discoidal complexes has come from the use of polarized attenuated total reflection infrared (ATR-IR) spectroscopy (Brasseur et al. (1990 Wald et al., 1990). Although internal reflection spectroscopy is not new (Harrick, 1967), its combination with polarized IR measurements of oriented biological membranes is a more recent application, which allows the spectrum to be taken in the presence of water. The technique has been used most frequently to study the orientation of lipid functional groups relative to the membrane plane in pure lipid bilayers (Fringeli and Gunthard, 1981 Holmgre et al., 1987 Okamura et al., 1990 Hubner and Mantsch, 1991), but has also been applied to studies of... 

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