Correlated spectroscopy processing

Fluorescence correlation spectroscopy (FCS) measures rates of diffusion, chemical reaction, and other dynamic processes of fluorescent molecules. These rates are deduced from measurements of fluorescence fluctuations that arise as molecules with specific fluorescence properties enter or leave an open sample volume by diffusion, by undergoing a chemical reaction, or by other transport or reaction processes. Studies of unfolded proteins benefit from the fact that FCS can provide information about rates of protein conformational change both by a direct readout from conformation-dependent fluorescence changes and by changes in diffusion coefficient. 

Other forms of spectrophotometric processes rely on Raman scattering. Raman scattering will now be briefly described, before our area of particular research interest, correlation spectroscopy, is discussed in detail. 

Particles of a size of less than 2 turn are of particular interest in Process Engineering because of their large specific surface and colloidal properties, as discussed in Section 5.2. The diffusive velocities of such particles are significant in comparison with their settling velocities. Provided that the particles scatter light, dynamic light scattering techniques, such as photon correlation spectroscopy (PCS), may be used to provide information about particle diffusion. 

NMR spectroscopy was carried out using a Varian Unity 300MHz spectrometer. Peptides were dissolved in 500 pL of 90% H,O/10% D20 (or 100% D20) giving a sample concentration of 1-2 mM and the pH adjusted to 5.5. H DQF-COSY (double quantum filtered two-dimensional correlated spectroscopy), ROESY, and TOCSY spectra were collected at 25 °C and processed as described.1 6-281... 

Dynamic processes at thermodynamic equilibrium that occur within a time range from sub-microseconds to seconds can be probed without the imposition of a transient disturbance by optical intensity fluctuation spectroscopy. As such, dynamic light scattering (DLS) [155] measures the fluctuation of quasielastic scattering intensity and fluorescence correlation spectroscopy (FCS) [156-158] measures concentration fluctuations of specific fluorescent molecules... 

The additional advantage of CARS-CS over DLS and FCS is the spectral selectivity for individual chemical components in their native state, where fluorescent labeling is not desired. This may not only allow mapping of 3D diffusion coefficients, for example inside life cells, but also offer a method to monitor the specific interaction of individual components within complex systems, e.g., aggregation processes of different chemical species. Another prospect is the implementation of CARS cross-correlation spectroscopy that may allow the investigation of correlated fluctuations between two different species. These could be two distinct Raman spectral features of one and the same compound, or a specific intrinsic Raman band and an emission of a more sensitive fluorescence label [160]. 

We have investigated peptides whose structures were known beforehand from NMR or x-ray spectroscopy and related these structures to 2D-IR spectroscopy. Ultimately, one would like to deduce the structure of an unknown sample from a 2D-IR spectrum. In the case of 2D NMR spectroscopy, two different phenomena are actually needed to determine peptide structures. Essentially, correlation spectroscopy (COSY) is utilized in a first step to assign protons that are adjacent in the chemical structure of the peptide so that J coupling gives rise to cross peaks in these 2D spectra. However, this through-bond effect cannot be directly related to the three-dimensional structure of the sample, since that would require quantum chemistry calculations, which presently cannot be performed with sufficient accuracy. The nuclear Overhauser effect (NOE), which is an incoherent population transfer process and has a simple distance dependence, is used as an additional piece of information in order to measure the distance in... 

Figure 15. Time constants of the a- and p-processes of several glass formers, as determined by dielectric spectroscopy (DS), light scattering (LS), photon correlation spectroscopy (PCS), NMR, Kerr effect (KE), neutron scattering (NS), and viscosity o-Terphenyl (OTP, type A) NMR (crosses, [177-179]), DS (filled squares [151]), KE (unfilled circles [66]), viscosity (solid line [164]). m-Tricresyl phosphate (m-TCP, type A) NMR (crosses [15]), LS (unfilled squares [181]), DS (circles [180]) and viscosity (line [182]). m-Fluoroaniline (FAN, type B) DS (stars [153]). 2-Picoline (PIC, type A) LS (unfilled circles [183]), NS (filled triangles [184]), PCS (unfilled squares [65], DS (filled diamonds, [181]). Toluene (type B) NMR (+ [11]), DS (filled squares [153]) and LS (filled circles [185]).

Other strategies that show great promise in reducing NMR acquisition time utilise methods to obtain multiple sets of data from one experiment through a concept known as time-shared evolution. An example of this process that should find utility in natural products elucidation was demonstrated by a pulse sequence called CN-HMBC.93 Traditionally, a separate 13C-HMBC and 15N-HMBC were acquired independently. However, the CN-HMBC allows both 13C- and 15N-HMBC spectra to be obtained simultaneously. By acquiring both data sets simultaneously, an effective 50% time reduction can be achieved.93 This approach has also been demonstrated for a sensitivity-enhanced 2D HSQC-TOCSY (heteronuclear multiple bond correlation total correlation spectroscopy) and HSQMBC (heteronuclear single quantum... 

These processes must be monitored to confirm that the second polymer does not emerge as a separate particle type. Re-nucleation, producing a crop of new particles, may be detected by progressive determination of particle size and comparing actual with the theoretical size calculated on the basis of constant particle number. This is easier to do in those processes where the monomer for the second polymer is added slowly at a steady and known rate and samples can be taken at regular time intervals for particle-size determination by electron microscopy, photon correlation spectroscopy or by disc centrifuge photo-sediometry. For particles prepared by non-aqueous dispersion... 

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