Fluorescence reabsorption

Reabsorption is a very important error source in thick scattering layers. The fluorescence is suppressed in the region of overlap with the absorption spectrum of 

Fi gure 8.20. Fluorescence reabsorption in a semi-infinite scattering layer. Ordinate fraction of emitted to true fluorescence intensity. Abscissa diffuse reflectance at the fluorescence wavelength. Parameter diffuse reflectance at the absorption wavelength, The scattering coefficient is assumed to be independent of A (S° = S ). 

In the semi-infinite layer approximation (Fb = F OI) the integral fluorescence quantum yield 

A few materials are given that can be used as primary fluorescence standards in the adsorbed state, because they have high yields, show little tendency of aggregation provided the surface coverage is low, and are photochemically stable  

In addition, the pure solid (N(C2Hs)4)2MnBr4 is recommended as standard. Its phosphorescence yield is high, j , = 0.8, the material has only medium absorption despite its high concentration, and there is no long-distance impurity quenching because the luminescence centers are electronically isolated from one another by the large counter ions. 

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From a practical point of view the consequences of TOF dispersion are important only for short intrinsic fluorescence decay times of to < 1 nsec. Figure 8.15 shows an example with to = 50 psec and realistic optical constants of the substrate. The intensity maximum in Fb(t) is formed at At 30 psec after (5-excitation. After this maximum, the fluorescence decays with an effective lifetime of r ff = 100 psec that increases after long times to t > > 500 psec. The long-lived tail disappears as soon as there is some fluorescence reabsorption, and for Ke = K there is practically no difference to the intrinsic decay curve (curve 3 in Figure 8.15). 

Figure 8.21. Fluorescence spectra of fluorescein on an alumina-TLC plate. Upper redshift of the spectra with concentration according to increasing fluorescence reabsorption. Lower corrected fluorescence spectrum of the most concentrated sample (dashed line) according in Figure 8.20.

Figure 8.22. Fluorescence reabsorption in a finite scattering layer with K°/S = 0.1 as a function of the scattering thickness Sd. Ordinate fraction of emitted to true fluorescence intensity in backward (upper) and forward direction (lower). Parameter reduced absorption coefficient at the fluorescence wavelength.

To quantify the concentration of a colorant, one must consider that linearity between the colorant concentration and the fluorescence emission intensity exists only at very low concentrations. The reason for deviation from linearity may be reabsorption of the emission light by other fluorophores or formation of dimers. If no extraction and controlled dilution of the fluorescent colorant are performed, the colorant quantification will be only qualitative. 

Another important linear parameter is the excitation anisotropy function, which is used to determine the spectral positions of the optical transitions and the relative orientation of the transition dipole moments. These measurements can be provided in most commercially available spectrofluorometers and require the use of viscous solvents and low concentrations (cM 1 pM) to avoid depolarization of the fluorescence due to molecular reorientations and reabsorption. The anisotropy value for a given excitation wavelength 1 can be calculated as... 

If the energy is transferred by trivial emission/reabsorption, it will lengthen the measured lifetime of the donor emission, not shorten it as happens in resonance energy transfer. This comes about because intervening absorption and emission processes take place prior to the final fluorescence emission (the reabsorption cannot take place until the photon has been emitted) the two processes do not compete dynamically, but follow in a serial fashion. In FRET, such an emission/reabsorption process does not occur, and the fluorescence lifetime of the donor decreases. This is an experimental check for reabsorption/reemission. 

Gorsuch and Hercules 109> stated that certain discrepancies between the fluorescence spectrum of 3-amino-phthalate dianion and the chemiluminescence spectrum of luminol are partly due to reabsorption of the shorter-wavelength chemiluminescence light by the luminol monoanion. These authors confirmed the results of E. H. White and M. M. Bursey 114> concerning the very essential solvent effect on luminol chemiluminescence the relative intensity of the latter in anhydrous DMSO/t-BuOK/ oxygen was found to be about 30,000 times that in DMSO/28 mole % water/potassium hydroxide/oxygen. 

Figure 8.2 presents the fluorescence of pyrene on silica gel. The loading is low so that pyrene is predominantly adsorbed as nonaggregated monomers (Mi). The backward fluorescence spectrum Fb of this sample is very comparable to the spectrum in polar solvents and not distorted by reabsorption. However, the forward spectrum Ft is almost completely suppressed in the region of overlap with the o -transition and hot sidebands of the weak first absorption band Si. The absorption coefficients of the sample vary widely from k" = 0.1 cm 1 (Si-band, Aa = 350-370 nm) to k = 25 cm-1 (S2-band, 1 290-340 nm), and in a first approximation the excitation spectrum of Fh reflects this variation correctly (Figure 8.2, left). The Ff-excitation spectrum, however, has only little in common with the real absorption spectrum of the sample. 

The total emitted fluorescence radiation without any reabsorption is then... 

An apparent decrease in the emission quantum yield and/or distortion of the band shape due to the reabsorption of emitted radiation. If such an effect is not corrected or compensated for, results of an emission experiment may prove to be incorrect. This is especially true in fluorescence quenching experiments conducted to evaluate the stoichiometry and affinity of ligand binding. 2. In a light irradiation experiment, the absorption of incident radiation by a species or molecular entity other than the intended primary absorber. See Fluorescence... 

Here (A) and crp (A) are the cross-sections for absorption and stimulated fluorescence at A, respectively, and mo is the population of the ground state. The first exponential term gives the attenuation due to reabsorption of the fluorescence by the long-wavelength tail of the absorption band. The attenuation becomes more important, the greater the overlap between the absorption and fluorescence bands. The cross-section for stimulated fluorescence is related to the Einstein coefficient by... 

It is called trivial because it does not require any energetic interaction between the donor and the acceptor. It is merely reabsorption of fluorescence radiation in accordance with Beer s Law and shows r 2 dependence on donor-acceptor distance. Although called trivial, it causes radiation imprisonment and can be important factor to be considered in fluorescence measurements. It may introduce error and distort emission spectrum by absorbing only that portion which overlaps its absorption spectrum. It is specially troublesome in studies on concentration quenching. 

Figure 28.15 a, Chemiluminescence spectrum obtained from electrolysis of a DMF solution containing 1 mM fluoranthene and 1 mAf 10-MP. Alternating steps at-1.75 V and +0.88 V vs. SCE were used, b, Chemiluminescence spectrum under the same conditions, but with 1 mM anthracene added. Inset shows anthracene fluorescence spectrum for a 10-5 M DMF solution. Reabsorption reduces the 0,0 intensity in b. [From Ref. 99, adapted with permission.]... 

In all cases the absorbance of the suspensions at 445 nm was smaller than 0.2. This small absorbance value minimizes reabsorption of the emitted light. Oxygen was not removed from the suspensions, since no measurable effect on the fluorescence intensity was found. 

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