Isoemissive point

There are many possibilities to use these complex formations in fluorescence sensing. If the excimer is not formed, we observe emission of the monomer only, and upon its formation there appears characteristic emission of the excimer. We just need to make a sensor, in which its free and target-bound forms differ in the ability of reporter dye to form excimers and the fluorescence spectra will report on the sensing event. Since we will observe transition between two spectroscopic forms, the analyte binding will result in increase in intensity of one of the forms and decrease of the other form with the observation of isoemissive point [22]. 

The isoemissive point is indicative of the involvement of only two species in the observed fluorescence. 

If definite stoichiometry is maintained in the exciplex formation, an isoemissive point similar to isosbestic point in absorption miy be observed. An interesting example of intra-molecular exciplex formation has been reported foi 9-methoxy-10-phenanthrenecarboxanil. The aniline group is not necessarily coplanar with the phenanthrene moiety but is oriented perpendicular to it. The u-elcctron located on its N-atom interacts with the excited -electron system and an intramolecular exciplex with T-bone type structure is formed in rigid glassy medium where rotation is restricted. Temperature dependence of fluorescence of this compound in methylcyclohexane-isopentane (3 1) solvent shows a definite isoemissive point (Figure 6.8). As the solvent melts and movement is restored to the molecule, structured fluorescence reappears. 

Figure 6.8 Temperature dependence of fluorescence emission from 9-methoxy-10-phenanthrenecarboxanil. Exciplex emission and isoemissive point.

Figure 3. Variation in the Eu emission spectrum for [Eu.2c] (pH 7.4, 0.1M MOPS) following incremental addition of sodium hydrogencarbonate spectra in the insets show formation of isoemissive points at 588 and 702 nm.

Titration of a constant concentration of BSA with TNS yields a decrease in the fluorescence intensity of the Trp residues together with an increase in the fluorescence intensity of TNS (not shown). We do not observe an isoemissive point at either pH, thus indicating that TNS has more than one binding site on BSA. After correcting the fluorescence intensities at the emission peak of Trp residues for the dilution and then for the optical densities at the excitation (Xex = 280 nm) and emission wavelengths (335 or 325 nm), we can plot the fluorescence intensity as a function of TNS concentration at pH 3 and 7 (Figure 15.3). Results indicate that the fluorescence-intensity decrease is more important at pH 3 than at pH 7. This means that the interaction between TNS and BSA is more important at a low pH. 

Fig. 2. The isosbestic points at 446 and 556 nm in the absorption spectra are matched by an isoemissive point at 685 nm indicating only two species present in solution, both of which are emissive. The shift in emission maximum from 606 nm in neutral solutions to 728 nm upon addition of acid may have interesting sensor applications. The results for 4 stand in contrast with results from dppz-containing Ru(II) tris diimine complexes, where dppz = dipyrido-ipyridophenazine, in which reversible protonation of quinoxaline N atoms leads to quenching of emission. Luminescence in frozen solvent glasses for 4 at 77 K is much stronger ( = 0.044 for the qdt complex), but still broad and without resolved structure.

Simultaneously, an isosbestic point at 625 nm and an isoemissive point at 662 nm are observed. These spectral results give positive evidence that the /3-emission is indeed from the excited state of the... 

Further bathochromic shifts on the Xmax and Xp and further increase in Ip are observed at [CHCl3]>.2.24 M. Absorption curves and emission curves no longer pass through their isosbestic or isoemissive points. This observation can either be attributed to the preferred solvation of the solute-solvent complex by chloroform as the concentration of chloroform increases or to the formation of a 1 n solute-solvent complex. The occurrence of these two events is presumably due to the highly localized concentration of chloroform in the solvation shell of squaraine, a consequence of the solute-solvent complexation process. 

We now decided to extend our measurements to low temperatures, in the hope of slowing down excitation transport to an accessible time regime. Emission spectra for pure polystyrene at low temperatures are shown in Figure 6. At the lowest temperatures a very strong monomer-like emission is evident. As the temperature is increased, this emission rapidly decreases, while excimer emission increases, resulting in a clean isoemissive point at 327 nm (13). 

At 150K, nearly all monomer-like emission has HTsappeared and the significant change above this temperature is that the excimer emission slightly broadens with loss of the isoemissive point. 

The solute-solvent complex model proposed above is further supported by results obtained from mixed-solvent experiments. In diethyl ether, Sq4 exists primarily as free squaraine (Fig. 3a). The addition of a complexing solvent should drive the equilibrium for complexation. As a result, both and Xp should shift to the red and the intensity of the p-emission should increase. To eliminate any complication due to the increase in dielectric constant of the mixed solvent during experimentation, the mixed-solvent experiment was first performed in a ternary system consisting of ether (e = 4.43), chloroform (e = 4.7), and n-hexane (e = 1.9). The addition of n-hexane in the mixture is to keep the dielectric constant constant as the concentration of chloroform increases. The absorption and fluorescence spectral results are summarized in Figs. 4a and 4b, respectively. The data showed that nd Xp shift to the red and the intensity of the P-emission increases as [CHCI3] increases at [CHCl3]<1.12 M. Simultaneously, an isosbestic point at -625 nm and isoemissive point -662 nm are observed in the absorption and fluorescence spectra, respectively. The observation of an isosbestic point in the absorption spectra and an isoemissive point in the emission spectra provide positive evidence that (1) Sq4 forms a complex with chloroform in the ethereal solutions,... 

In PBS buffer, free calcofluor white displays a fluorescence that is modified in presence of ai-acid glycoprotein Xq =295 nm). Figure 8.37 shows emission spectra of a fixed amount of a i-acid glycoprotein before and after addition of variable amounts of calcofluor white, the excitation wavelength being 295 nm. As the calcofluor concentration increases, its emission around 450 nm increases, while emission of the Trp residues (at 333 nm) decreases. An isoemissive point is observed at 395 nm. The analysis of the decrease of the fluorescence intensity of the Trp residues yields a stoichiometry for the protein-calcofluor complex equal to 1 (see figure 8.11). 

Figure 11.7 Time-resolved emission spectra ofJCS in ethanol at 298 K reconstructed from emission decay curves. An isoemissive point is observed at540nm (18520cm ) [23]. (Reproduced with permission.)...

Figure 2. Steady-state fluorescence spectra of HPTS in water at room temperature [13]. Curve labelled 0 is at neutral pH. Other spectra are at low pH and are labelled by the volume fraction of 70% perchloric acid added. Note the isoemissive point at 488nm.

Figure 4,7 Molecular structure and temperature dependence of (a, b) FSF and (c, d) FASAF emission spectra in ethanol. Dual emission is observed with isoemissive points below 230 K for both compounds. Adapted with permission from Figure 6 of F. B. Dias, 5. Pollock, C. Hedley, L. O. Palsson, Andy Monkman, 1.1. Perepichka, I. F. Perepichka, M. Tavasli and M. R. Bryce, Intramolecular charge transfer assisted by conformational changes in the excited state of fluorene-dibenzothiophene-S,S-dioxide co-oligomers, J. Phys. Chem. B, 110, 19329 19339 (2006). Copyright 2006 American Chemical Society...

T is a constant over the range of temperature investigated. This assumption has often been used when the temperature-dependent fluorescence spectra are characterized by an isoemissive point. In somes cases, is temperature-dependent despite the existence of the isoemissive point (2). Thus, taking the 1 / ratio as proportional to may lead to incorrect conclusions aDOut the matrix mobility. Only transient measurements allow a quantitative determination of the dynamic properties of the probe. 

Fig. 4 gives evidence of the temperature dependence of the excimer lifetime of diphant even in the range of existence of the isoemissive point. But contrary to T, x Plotted versus temperature exhibits little scatter within the range of polymers studied. The similar values obtained in solution (5) show that X is unaffected by the rigidity of the matrix and is, indeed, and intrinsic characteristic of the fluorescence probe. 

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