Fluorometry, steady-state

Dr can be determined by time-resolved fluorescence polarization measurements, either by pulse fluorometry from the recorded decays of the polarized components I l and 11, or by phase fluorometry from the variations in the phase shift between J and I as a function of frequency (see Chapter 6). If the excited-state lifetime is unique and determined separately, steady-state anisotropy measurements allow us to determine Dr from the following equation, which results from Eqs (5.10) and (5.41) ... 

The helium-neon (HeNe) laser immediately comes to mind, having a very useful spectral line at 633 nm for steady-state red/near-IR fluorescence studies. Kessler and Wolfbeis have demonstrated the fluorescence assay of the protein human serum albumin using the probe albumin blue excited with a red HeNe laser.(71) Another useful wavelength available from the green HeNe laser is at 543.5 nm and this has been used with phase-modulation fluorometry by Lakowicz etal. to study probes such as carboxy seminaphtorhodafluor-6 (SNARF-6) as a means of measuring pH.(72)... 

The performance of the R928 and its variants has been well documented in pulse 12,31,78 81) and phase-modulation 57, 82) lifetime fluorometry. The lower cost and gain of side-window photomultipliers makes them the best all-round compromise for use in dual purpose instruments with both steady-state and lifetime capabilities. 

The elucidation of the intramolecular dynamics of tryptophan residues became possible due to anisotropy studies with nanosecond time resolution. Two approaches have been taken direct observation of the anisotropy kinetics on the nanosecond time scale using time-resolved(28) or frequency-domain fluorometry, and studies of steady-state anisotropy for xFvarying within wide ranges (lifetime-resolved anisotropy). The latter approach involves the application of collisional quenchers, oxygen(29,71) or acrylamide.(30) The shortening of xF by the quencher decreases the mean time available for rotations of aromatic groups prior to emission. 

It is well known that both nanometre and nanosecond-picosecond resolutions at an interface can be achieved by total internal reflection (TIR) fluorescence spectroscopy. Unlike steady-state fluorescence spectroscopy, fluorescence dynamics is highly sensitive to microscopic environments, so that time-resolved TIR fluorometry at water/oil interfaces is worth exploring to obtain a clearer picture of the interfacial phenomena [1]. One of the interesting targets to be studied is the characteristics of dynamic motions of a molecule adsorbed on a water/oil interface. Dynamic molecular motions at a liquid/liquid interface are considered to be influenced by subtle changes in the chemical/physical properties of the interface, particularly in a nanosecond-picosecond time regime. Therefore, time-resolved spectroscopy is expected to be useful to study the nature of a water/oil interface. 

In the first section, steady-state spectroscopy is used to determine the stoichiometry and association constants of molecular ensembles, emphasize the changes due to light irradiation and provide information on the existence of photoinduced processes. Investigation of the dynamics of photoinduced processes, i.e. the determination of the rate constants for these processes, is best done with time-resolved techniques aiming at determining the temporal evolution of absorbance or fluorescence intensity (or anisotropy). The principles of these techniques (pulse fluorometry, phase-modulation fluorometry, transient absorption spectroscopy) will be described, and in each case pertinent examples of applications in the flelds of supramolecular photophysics and photochemistry will be presented. 

The various examples of photoresponsive supramolecular systems that have been described in this chapter illustrate how these systems can be characterized by steady-state and time-resolved spectroscopic techniques based on either absorption or emission of light. Pertinent use of steady-state methods can provide important information in a simple vay stoichiometry and stability constant(s) of host-guest complexes, evidence for the existence of photoinduced processes such as electron transfer, energy transfer, excimer formation, etc. Investigation of the dynamics of these processes and characterization of reaction intermediates requires in most cases time-resolved techniques. Time-resolved fluorometry and transient absorption spectroscopy are frequently complementary, as illustrated by the study of photoinduced electron transfer processes. Time-resolved fluorometry is restricted to phenomena whose duration is of the same order of magnitude as the lifetime of the excited state of the fluorophores, whereas transient absorption spectroscopy allows one to monitor longer processes such as diffusion-controlled binding. 

The deprotonation of carbazole, 48, at pH = 12 was studied by steady-state and time-resolved fluorometry. It was found that 5 x 10 M y-CD... 

Two-dimensional steady-state fluorometry can therefore be applied for the characterization of different water sources, such as spring water, surface and groundwater supplies and wastewater streams. As can be easily seen in Figure 12.7, different water streams originate rather different fluorescence maps, where it is possible to identify multiple fluorescence regions and, ultimately, associate some of these regions with the presence of defined constituents. 

The combined use of different natural fluorescence techniques, such as steady-state fluorometry, fluorescence anisotropy and time-decay fluorescence, has been revealed to be quite powerfiil. The use of these techniques in an integrated mode for the monitoring of membrane-protein interactions is only in its infancy. These techniques offer not only the possibility to study the interaction of proteins with membranes, under convective and diffusive conditions, but also they may be easily extended to studies involving proteins and other porous materials such as chromatography media. The areas of application of these techniques will range from polypeptide and protein fractionation to the monitoring of hemodialysis systems. 

Fluorescence measurements and detection can either be made under steady-state or time-resolved conditions. Some of the commonly used measurement techniques focus on changes in optical properties such as fluorescence intensity quenching, phase fluorometry (lifetime), polarization, surface plasmon resonance (SPR), and evanescent waves. Here, we will present detector systems based for (a) fluorescence intensity quenching and (b) phase fluorometry in detail. A few example references of integrated optical sensor systems based on the various optical measurement techniques are given in Table 1 and the reader is encourage to review those papers if more details are desired. 

This ratio is an indication of the structural order of the fluid matrix, and for an isotropic fluid it should be close to unity [4]. In order to differentiate between anisotropy of the medium and that of the molecular rotations, one should compare the V vs. To plots (determined from the steady-state anisotropy measurements at low temperatures in high density fluids at different excitation wavelengths) for a particular fluorophore embedded in an isotropic medium to a similar plot for the probe embedded in the test fluid. If, for example, a strong dependence of v on the value of ro is evident for both media and the ratio of in-plane to out-of-plane rotational rates is very high (>10), one can conclude that the rotations are anisotropic but the medium is isotropic. The technique of differential polarized phase fluorometry [8,12], which is beyond the scope of this chapter, has been successfully applied to study the types of rotations displayed by fluorophores embedded in different media. 

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