Steady-state spectra

In this section we will review the application of near-IR system instrumentation to the most commonly encountered fluorescence measurements such as steady-state spectra, excited state lifetimes, anisotropy, microscopy, multiplexing, high-performance liquid chromatography (HPLC), and sensors. 

The first measurement we make when starting a fluorescence study is not usually a fluorescence measurement at all but the determination of the sample s absorption spectrum. Dual-beam differential spectrophotometers which can record up to 3 absorbance units with a spectral range of 200-1100 nm are now readily available at low cost in comparison to fluorimeters. The wide spectral response of silicon photodiode detectors has made them preeminent over photomultipliers in this area with scan speeds of a few tens of seconds over the whole spectral range being achieved, even without the use of diode array detection. 

Background fluorescence from glass cut-off filters is generally much less when exciting in the near-IR as compared with the UV/visible. Interference filters for the near-IR are also readily available. 

Andrews-Wilberforce and Patonay(10) have studied the steady-state metal fluorescence quenching of a series of carbocyanine laser dyes in methanol and listed the peak absorption and emission wavelengths of the dyes. Table 12.1 summarizes the spectral properties of the dyes studied. The small Stokes shift shown in Table 12.1 highlights 

Until recently there were comparatively few reports of fluorescence lifetime studies of dye molecules in the near-IR, but this situation has changed rapidly. The fluorescence lifetimes of near-IR emitting dyes such as carbocyanines, porphyrins, oxazines, and xanthenes, are usually in the nanosecond region, consistent with the high oscillator strength of the Si-So transition in such compounds. 

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For excitation of solutes with 0-0 transitions v0o>v (antiStokes spectral region of absorption), the situation is the opposite at the initial instant of time, the spectra are red-shifted as compared to the steady state spectra, Av1 (l)<0. In this case, the return of the spectrum to its normal position during configurational relaxation will lead to a blue shift with time. From the physical point of view, this means that the intermolecular energy excess, which the solvates possess before excitation, is partially converted into emitted energy leading to an increase in the radiation frequency with time. That is why the process may be called the up-relaxation of the fluorescence spectra. 

The role of the conditions in which these phenomena are observed is now well understood [40, 45], The chromophore should be solvatofluorochromic, that is, its fluorescence spectra should respond to changes in interaction energy with its environment by significant shifts. This environment should be relatively polar, but rigid or highly viscous, so that the relaxation times of its dipoles, tr, are comparable or longer than the fluorescence lifetime tf (in the case of recording the steady-state spectra) or on the time scale of observation (in time-resolved spectroscopy). Thus, these effects are coupled with molecular dynamics in condensed media. 

Figure 12.1 shows the classic L-format of the most commonly used fluorescence spectrometer configuration which is topologically the same for the measurement of both steady-state spectra and lifetimes. The source and detector options of relevance to IR fluorescence measurements are discussed in Sections 12.3 and 12.4, respectively. The other optical components comprised of the lenses for focusing and collection and monochromators for wavelength selection contain few peculiarities in the near-IR as... 

This equation is a good approximation to the description of the relaxa-tional spectral shifts occurring with variations of xR and xF, which are brought about by temperature changes and effects of collisional fluorescence quenchers. Using this equation, xR can be easily determined if xF, v0, and vs are known for the system (the chromophore and its environment) under study. The last two values may be obtained not only from time-resolved spectra but also from steady-state spectra at the lowest (v0)and highest (vE) temperatures. The latter measurement is difficult to achieve with such labile... 

Table 1.3. Luminescence centers found in steady-state spectra of minerals ...

The luminescence centers Eu ", Yb, Ce, Dy, and Sm + characterize the steady-state spectra of danburite (Gaft et al. 1979 Gaft 1989). By using laser-induced time-resolved spectroscopy we were able to detect the following emission centers Ce, Eu, Eu, Sm ", Dy (Fig. 4.15)... 

Two different Mn " luminescence centers have been found in steady-state spectra of apophyllite in the Ca position with orange luminescence peaking at 620 nm and in the K position with green emission peaking at 500 nm (Tarashchan 1978). The apophyllite in our study consisted of three samples from different environments. The laser-induced time-resolved technique enables us to detect the following emission centers Ce ", Mn " " with orange emission and possibly (U02) (Fig. 4.19). 

The structure of charoite is monocUnic-prismatic (2/m) with space group P /4. Luminescence centers Ce, Eu + and Mn + characterize the steady-state spectra of charoite (Gaft 1989 Gorobets and Rogojine 2001). 

Fig. 4.50. Laser-induced time-resolved luminescence spectra of ruby (a) and steady-state spectra of sapphire (b) demonstrating Cr and possibly Fe centers...

The structure of boehmite contains double sheets of octahedra with A1 ions at their centers, and the sheets themselves are composed of chains of octahedra. In diaspor the oxygens are in a hexagonal close packed layer those within the double octahedral layers in boehmite are in a cubic pacldng relationship. Luminescence center Cr characterizes steady-state spectra (Solomonov et al. 1994 Shoval et al. 1999). The natural boehmite and diaspor in our study consisted of twelve samples. The laser-induced time-resolved technique enables us to detect Cr emission centers (Figs. 4.62-4.63). 

Thorite and orangite (orange thorite) have a tetragonal structure and are isostructural with zircon. Steady-state spectra under X-ray and laser (337 nm) excitations are connected with REE " ", namely Sm " ", Tb ", Dy " " and Eu ". Reabsorption lines of Nd " have been also detected (Gorobets and Rogojine 2001). Laser-induced time-resolved luminescence enables us to detect Eu " and uranyl emission centers (Eig. 4.70). 

Narrow bands at 320-335 nm with very short decay time of 20-30 ns may be confidently ascribed to Ce luminescence (Fig. 4.44). In steady-state spectra different bands in this spectral range without decay time analyses, especially under X-ray and electron beam excitations may be mistakenly considered as Ce " emissions (GOtze 2000). 

Eigures 5.14a,b represent luminescence spectra of scheelite enriched by Eu. Luminescence of Eu " is well known in steady-state spectra of scheelite (Tarash-chan 1978 Gorobets and Kudrina 1980). In time-resolved spectroscopy its relative intensity is stronger after a long delay time, which is explained by the longest decay time of Eu " in scheelite compared to other REE. 

The LIBS technique may be extremely useful for sorting of fluorite ores. Figure 8.8 clearly demonstrates the opportunities of time-resolved LIBS in comparison with the steady-state method in the case of fluorite-carbonate ores. Fluorite and calcite both has Ca as a major element and its emission lines dominate in the steady-state spectra making sorting impossible. After a delay of several ps the intensity of Ca lines is strongly diminished and a F line with a longer decay becomes visible in the fluorite spectrum. 

The steady state spectra of indole solvated in water show similar behavior as those from indole solvated in ethanol (see Fig. la)). Due to the larger dipole moment of the water molecules a somewhat larger Stokes shift of the fluorescence spectrum is observed. 

Fig. 1-left gives a general overview of the differential absorption spectra recorded for the free chromophore, oxyblepharismin, dissolved in DMSO for reference the steady-state absorption and (uncorrected) fluorescence spectra are also given below, in dotted lines. At all pump-probe delay times, the overall picture is a superposition of the structured bleaching and gain bands, as expected from the steady-state spectra, and broad transient absorption bands around 530 nm and 750 nm (weaker). These apparently homothetic spectra are very similar to... 

The steady-state spectra obtained for different alcohols are depicted in fig. 1. While the absorption spectra red shift with increasing solvent polarisability (from methanol to octanol), the fluorescence shows a red-shift when going from octanol to methanol. The total Stokes shifts are very large 7.900 100 cm 1 for PSBR/MeOH and 6.870 100 cm 1 for octanol. Another striking observation is the 30 % smaller width of the fluorescence spectrum of methanol (AE = 3.420 cm 1) compared with other alcohols. While the widths of the fluorescence spectra are solvent-dependent, the absorption spectra have a FWHM of -5.100 cm"1, irrespective of the solvent. As we will substantiate in the following, this behavior indicates that the potential energy surface around the fluorescent point is different than near the Franck-Condon zone probed by absorption, as suggested by quantum chemistry calculations [7]. 

To study the excited state one may use transient absorption or time-resolved fluorescence techniques. In both cases, DNA poses many problems. Its steady-state spectra are situated in the near ultraviolet spectral region which is not easily accessible by standard spectroscopic methods. Moreover, DNA and its constituents are characterised by extremely low fluorescence quantum yields (<10 4) which renders fluorescence studies particularly difficult. Based on steady-state measurements, it was estimated that the excited state lifetimes of the monomeric constituents are very short, about a picosecond [1]. Indeed, such an ultrafast deactivation of their excited states may reduce their reactivity something which has been referred to as a "natural protection against photodamage. To what extent the situation is the same for the polymeric DNA molecule is not clear, but longer excited state lifetimes on the nanosecond time scale, possibly of excimer like origin, have been reported [2-4],... 

Upon adsorption on Zr02 the amount of water molecules in the first solvation shell of C343 is decreased due to the presence of the surface. The reduced solvation shell as well as prealignement of the water molecules around the Zr02 surface is probably behind the reduction of the Stokes shift which is observed in the steady state spectra. 

However, a study of a few dyes of higher fluorescence quantum yield in polymer microparticles did not show any change in the fluorescence lifetime even though the modification of the fluorescence spectra was observed [4]. In this work, a new molecule (9-amino acridine hydrochloride hydrate, 9AAHH) is reported in which we have observed the effect of MDR in both, the steady state spectra and the fluorescence lifetimes. The dephasing time of 9AAHH in polymer matrix at room temperature have been determined from this study. 

It can be asked why the esters exhibit this increased tendency toward the TICT state. The answer from steady-state spectra is that although A increases for the esters by a factor of roughly 2, the main reason is a strong increase of the TICT formation rate kBA, 33... 

Figure 1 illustrates typical time-resolved emission spectra of C153 observed in the solvents acetonitrile, chloroform, and 1-pentanol. Also shown (dashed curves) are the steady-state emission spectra (equal to the time infinity spectra in most cases) and the estimated time-zero spectra, the spectra expected prior to any solvent relaxation. (The latter were obtained through comparison of steady-state spectra in the solvent of interest and in a non-polar reference solvent as described in Ref. 17.) A few features of these spectra are noteworthy. 

SCHEME 16.3 The dark and CIDNP steady state spectra indicated the formation of degradation prodncts. (Redrawn from Pahn, W.-U., and. Dreeskamp, H. J. Photochem Photobiol, A Chemistry, 52, 439-450, 1990 Pahn, W.-U., Dreeskamp, H., Castellan, A., and Bonas-Laurent, H., Ber. Bunsenges Phys. Chem., 96, 50-61, 1992.)... 

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