Depolarization spectroscopy

Characterization of Aquatic Humic Acid Fractions by Fluorescence Depolarization Spectroscopy... 

This chapter presents new information about the physical properties of humic acid fractions from the Okefenokee Swamp, Georgia. Specialized techniques of fluorescence depolarization spectroscopy and phase-shift fluorometry allow the nondestructive determination of molar volume and shape in aqueous solutions. The techniques also provide sufficient data to make a reliable estimate of the number of different fluorophores in the molecule their respective excitation and emission spectra, and their phase-resolved emission spectra. These measurements are possible even in instances where two fluorophores have nearly identical emission specta. The general theoretical background of each method is presented first, followed by the specific results of our measurements. Parts of the theoretical treatment of depolarization and phase-shift fluorometry given here are more fully expanded upon in (5,9-ll). Recent work and reviews of these techniques are given by Warner and McGown (72). 

Steady-State Fluorescence Depolarization Spectroscopy. For steady state depolarization measurements, the sample is excited with linearly polarized lig t of constant intensity. Observed values of P depend on the angle between the absorption and emission dipole moment vectors. In equation 2 (9), Po is the limiting value of polarization for a dilute solution of fluorophores randomly oriented in a rigid medium that permits no rotation and no energy transfer to other fluorophores ... 

The rotational reorientation times of the sample in several solvents at room temperature were measured by picosecond time-resolved fluorescence and absorption depolarization spectroscopy. Details of our experimental setups were described elsewhere. For the time-correlated single photon counting measurement of which the response time is a ut 40 ps, the sample solution was excited with a second harmonics of a femtosecond Ti sapphire laser (370 nm) and the fluorescence polarized parallel and perpendicular to the direction of the excitation pulse polarization as well as the magic angle one were monitored. The second harmonics of the rhodamine-640 dye laser (313 nm 10 ps FWHM) was used to raesisure the polarized transient absorption spectra. The synthesis of the sample is given elsewhere. All the solvents of spectro-grade were used without further purification. 

Analysis of experiments which monitor the rate of excitation transport among naphthyl chromophores in low concentration on isolated coils of poly-(2-vinylnaphthalene-co-methyl methacrylate) in poly-(methyl methacrylate) (PMMA) host by time-resolved fluorescence depolarization spectroscopy allows the quantitative detemination of the copolymer... 

The rapid decay of absorption of excited states on the ps time scale has been measured for pyrazolotriazole azomethine dyes. The molecular orientation dynamics of polar dye probes in t-butanol-water mixtures have been determined by ps fluorescence depolarization spectroscopy. Dyes studied in this investigation were the monocations nile blue and thionine, resorufin a monoanion, and nile red a polar but neutral molecule. A very detailed ps study of rotational diffusion of excited states of merocyanine-540 in polar solvents, has also been reported . 

Spontaneous depolarization spectroscopy is like TSPC except that no electric field is applied whatsoever during the entire course of the experiment. The electrometer measures any current generated in the sample as a result of dipolar motion over the temperature range of the experiment. This method can be used, for example, to study the internal stress frozen into a sample during processing. 

Luminescence, Excitation, and Depolarization Spectroscopy, and Measurement of Lifetimes... 

Dielectric Depolarization Spectroscopy Thermally Stimulated Current Technique... 

The next section, structure-rheology-texture relationships, deals with the quantification and modeling of the structure and the mechanical properties of lipid networks, and then examines cocoa butter and milkfat within this general framework. The chapter on milkfat focuses on the effects of the minor components contained in native milkfat on structure formation and final mechanical properties. This section also contains a chapter on the use of fluorescence depolarization spectroscopy as a tool to determine microviscosity and structural order in lipid systems and an overview of the texture of fat systems. 

The same information may be obtained from purely rotational far infrared spectroscopy (FIR) and depolarized Rayleigh spectra. Dielectric relaxation measurements are also used for the same goal, most successfully in combination with far-infrared data. The absorption coefficient of a periodic electric field... 

A review article has been published on depolarized Raman spectroscopy in molten salts, and it is discussed briefly here for the case of molten nitrates. Polarized (VV) and depolarized (HV) Raman scattering of the 1050 cm" line was used. The intensity of the isotropic part of the... 

Lipari G. and Szabo A. (1980) Effect of Vibrational Motion on Fluorescence Depolarization and Nuclear Magnetic Resonance Relaxation in Macromolecules and Membranes, Biophys. J. 30, 489—506. Steiner R. F. (1991) Fluorescence Anisotropy Theory and Applications, in Lakowicz J. R. (Ed.), Topics in Fluorescence Spectroscopy, Vol. 2, Principles, Plenum Press, New York, pp. 127-176. 

R. E. Dale, Membrane structure and dynamics by fluorescence probe depolarization kinetics, in Time-Resolved Fluorescence Spectroscopy in Biochemistry and Biology (R. B. Cundall and R. E. Dale, eds.), pp. 555-612, Plenum, New York (1984). 

Physico-chemical properties. In the fifties and sixties, several studies on the conformation of ACTH in solution were carried out. Among the used techniques were ORD, CD, fluorescence depolarization studies and kinetics of deuterium hydrogen exchange (for a review see ref. 2). The results pointed to a highly flexible random coil in solution however, Eisinger (40) found that the distance between Tyr and Trp [in ACTH-(1-24)] as measured by excitation spectroscopy, was in better agreement with some form of loop or helical structure. In addition. Squire and Bewley noted 11-15% helical content, located in the N-terminal 1-11 part of the molecule, when measuring the ORD of ACTH at pH 8.1 (41) (a random coil was found at neutral and acidic pH values, 2). 

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