Fluorescence intensity, linear spectroscopy

Like Raman scattering, fluorescence spectroscopy involves a two-photon process so that it can be used to determine the second and the fourth rank order parameters. In this technique, a chromophore, either covalently linked to the polymer chain or a probe incorporated at small concentrations, absorbs incident light and emits fluorescence. If the incident electric field is linearly polarized in the e direction and the fluorescent light is collected through an analyzer in the es direction, the fluorescence intensity is given by... 

After extraction, the urethanated films were subjected to alkaline hydrolysis of urethanes to liberate the corresponding amines, while the adipoylated films were hydrolyzed after having reacted with 7-hydroxycoumarin. Amounts of the released amines and coumarin were determined by fluorescence spectroscopy as described in the Experimental section. Since aniline as well as butylamine has no appreciable fluorescence by themselves, their fluorescence assay was made after reacting with o-phthalaldehyde in the presence of mercaptoethanol. In Figure 3, where relative fluorescence intensities are plotted as a function of concentrations of amines and hydroxycoumarin, one can see that the fluorescence intensities vary linearly with their concentration to permit us the quantitative determination of extremely small amounts of amines and hydroxycoumarin. 

The high sensitivity of fluorescence spectroscopy and the linear proportionality between fluorescence intensity and fluorophore concentration in diluted solutions is an asset, and usually makes fluorescence the technique of choice for studying host-pyrene interactions. Nonetheless, the smallest presence of fluorophore impurities may bias the emission spectra. This method is applicable for large K values (up to 10 M ). 

Fluorescence spectroscopy is a popular and highly sensitive technique (down to the nanomolar detection) for studying host-guest systems, and very large association constants (up to 10 M ) can be estimated by this method. At low concentration, the fluorescence intensity is linearly proportional to the concentration of the fluorophore. The observed fluorescence can be described by Eq. (13.9) ... 

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 ... 

An additional piece of information can be obtained by studying a synthetic compound derived from the GFP chromophore (1-28) fluorescing at room temperature. In Fig. 3a we show the chemical structure of the compound that we studied in dioxan solution by pump-probe spectroscopy. If we look at the differential transmission spectra displayed in Fig. 3b, we observed two important features a stimulated emission centered at 508 nm and a huge and broad induced absorption band (580-700 nm). Both contributions appear within our temporal resolution and display a linear behavior as a function of the pump intensity in the low fluences limit (<1 mJ/cm2). We note that the stimulated emission red shifts with two characteristic time-scales (500 fs and 10 ps) as expected in the case of solvation dynamics. We conclude that in the absence of ESPT this chromophore has the same qualitative dynamical behavior that we attribute to the relaxed anionic form. 

Raman spectroscopy has been widely used to study the composition and molecular structure of polymers [100, 101, 102, 103, 104]. Assessment of conformation, tacticity, orientation, chain bonds and crystallinity bands are quite well established. However, some difficulties have been found when analysing Raman data since the band intensities depend upon several factors, such as laser power and sample and instrument alignment, which are not dependent on the sample chemical properties. Raman spectra may show a non-linear base line to fluorescence (or incandescence in near infrared excited Raman spectra). Fluorescence is a strong light emission, which interferes with or totally swaps the weak Raman signal. It is therefore necessary to remove the effects of these variables. Several methods and mathematical artefacts have been used in order to remove the effects of fluorescence on the spectra [105, 106, 107]. 

Many features of the emission spectrum can show time dependence, including the spectral shape (l,3 (v-9), the peak intensity, the linear polarization (10) and, in principle, the circular polarization (11). In extreme cases, the emission spectrum can actually have two separate fluorescence bands from two different isomers of the electronically excited molecules (12-15). For molecules with this behavior, it is possible to determine the kinetics of excited state isomerization by transient emission spectroscopy. 

Because the atomic fluorescence is measured at a right angle to the source, spectral interferences are minimal and a simple cutoff filter may often be used to isolate the emission line. The intensity of the fluorescence is directly proportional to the analyte concentration. As the analyte concentration within the flame becomes large, self-absorption of resonance fluorescence becomes significant, as it does in flame emission spectroscopy. Under these conditions, the linearity of the instrumental response breaks down and a calibration curve must be used or the analyte solutions diluted accordingly. 

In excited-state spectroscopies, including fluorescence spectroscopy, spectroscopic intensity is usually linear in functions of each of three or more independent variables, so that a three-way array of data can be fit with a trilinear model. The presence of three or more linear relationships makes algebraic methods for resolving the spectra and other properties of individual components substantially more powerful than in the case of two linear relationships. The use of a general trilinear model is sometimes known as three-way factor analysis, three-mode factor analysis, or threemode principal component analysis. For a review of the mathematics and application to spectroscopy, see our survey article. ... 

This indicates that a linear relation exists between the intensity of fluorescence and the concentration of the fluorescing species and provides the theoretical basis for analytical atomic fluorescence spectroscopy. 

The VUV intensity generated by nonresonant conversion methods is sufficient for most investigations in linear (absorption or fluorescence) spectroscopy. Other applications (like multiphoton excitation and ionization or photodissociation) require more powerful light pulses. 

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