Emission spectrum lifetime

The interpretation of emission spectra is somewhat different but similar to that of absorption spectra. The intensity observed m a typical emission spectrum is a complicated fiinction of the excitation conditions which detennine the number of excited states produced, quenching processes which compete with emission, and the efficiency of the detection system. The quantities of theoretical interest which replace the integrated intensity of absorption spectroscopy are the rate constant for spontaneous emission and the related excited-state lifetime. 

Theory. If two or more fluorophores with different emission lifetimes contribute to the same broad, unresolved emission spectrum, their separate emission spectra often can be resolved by the technique of phase-resolved fluorometry. In this method the excitation light is modulated sinusoidally, usually in the radio-frequency range, and the emission is analyzed with a phase sensitive detector. The emission appears as a sinusoidally modulated signal, shifted in phase from the excitation modulation and partially demodulated by an amount dependent on the lifetime of the fluorophore excited state (5, Chapter 4). The detector phase can be adjusted to be exactly out-of-phase with the emission from any one fluorophore, so that the contribution to the total spectrum from that fluorophore is suppressed. For a sample with two fluorophores, suppressing the emission from one fluorophore leaves a spectrum caused only by the other, which then can be directly recorded. With more than two flurophores the problem is more complicated but a number of techniques for deconvoluting the complex emission curve have been developed making use of several modulation frequencies and measurement phase angles (79). 

Consider a sample with two fluorophores, A and B, whose lifetimes (lA and XB) are each independent of emission wavelength and are different fram one another. By setting 4>D = 4>A 90°, the contribution from A is nulled out and the scanned emission spectrum represents only the contribution from B. Similarly, the spectrum for A is obtained by setting 4>D = 4>B 90°. In practice, finding the correct value of for this... 

Fig. 3.6 (a) Decay scheme of and (b) ideal emission spectrum of Co diffused into rhodium metal. The nuclear levels in (a) are labeled with spin quantum numbers and lifetime. The dashed arrow up indicates the generation of Co by the reaction of Mn with accelerated deuterons (d in Y out). Line widths in (b) are arbitrarily set to be equal. The relative line intensities in (%) are given with respect to the 122-keV y-line. The weak line at 22 keV, marked with ( ), is an X-ray fluorescence line from rhodium and is specific for the actual source matrix... 

Energy transfer by the trivial mechanism is characterized by (a) change in the donor emission spectrum (inner filter effect), (b) invariance of the donor emission lifetime, and (c) lack of dependence upon viscosity of the medium. 

Finally, a nonfluorescent YFP variant has been constructed for use as a FRET acceptor for GFP [89]. This allows the detection of the complete emission spectrum of GFP, while the FRET efficiency is high (R0 = 5.9 nm) due to strong overlap of the GFP fluorescence and YFP absorbance band. The occurrence of FRET was detected by a reduction in the excited state lifetime of the GFP by FLIM. The main disadvantage is that the presence of the acceptor cannot be detected in living cells. 

Thus, E is defined as the product of the energy transfer rate constant, ku and the fluorescence lifetime, xDA, of the donor experiencing quenching by the acceptor. The other quantities in Eq. (12.1) are the DA separation, rDA the DA overlap integral, / the refractive index of the transfer medium, n the orientation factor, k2 the normalized (to unit area) donor emission spectrum, (2) the acceptor extinction coefficient, eA(k) and the unperturbed donor quantum yield, QD. 

Certain features of light emission processes have been alluded to in Sect. 4.4.1. Fluorescence is light emission between states of the same multiplicity, whereas phosphorescence refers to emission between states of different multiplicities. The Franck-Condon principle governs the emission processes, as it does the absorption process. Vibrational overlap determines the relative intensities of different subbands. In the upper electronic state, one expects a quick relaxation and, therefore, a thermal population distribution, in the liquid phase and in gases at not too low a pressure. Because of the combination of the Franck-Condon principle and fast vibrational relaxation, the emission spectrum is always red-shifted. Therefore, oscillator strengths obtained from absorption are not too useful in determining the emission intensity. The theoretical radiative lifetime in terms of the Einstein coefficient, r = A-1, or (EA,)-1 if several lower states are involved,... 

Experimentally, Eem is available from the emission spectrum and knr from a combination of excited state lifetime (x0) and emission quantum yield (<(>r) measurements as shown in eq. 15. An... 

Chemiluminescence is believed to arise from the 2Bj and the 2B2 electronic states, as discussed above for the reaction of NO with ozone [17]. The primary emission is in a continuum in the range =400-1400 nm, with a maximum at =615 nm at 1 torr. This emission is significantly blue-shifted with respect to chemiluminescence in the NO + 03 reaction (Xmax = 1200 nm), as shown in Figure 2, owing to the greater exothermicity available to excite the N02 product [52], At pressures above approximately 1 torr of 02, the chemiluminescence reaction becomes independent of pressure with a second-order rate coefficient of 6.4 X 10 17 cm3 molec-1 s-1. At lower pressures, however, this rate constant decreases and then levels off at a minimum of 4.2 X 1(T18 cm3 molec-1 s-1 near 1 mtorr, and the emission maximum blue shifts to =560 nm [52], These results are consistent with the above mechanism in which the fractional contribution of (N02 ) to the emission spectrum increases as the pressure is decreased, therefore decreasing the rate at which (N02 ) is deactivated to form N02. Additionally, the radiative lifetime and emission spectrum of excited-state N02 vary with pressure, as discussed above for the NO + 03 reaction [19-22],... 

The emission spectrum consists of a series of weak bands starting at about 220 nm and then growing into a continuum from about 240 to 400 nm, with a maximum at approximately 270 nm as shown in Figure 5. Halstead and Thrush estimated that =65% of the emission occurs from the B2 state, =15% from the 3B3, and =20% from a combination of the A2 and Bi states [24, 28, 29] with a rate constant of 2 X 1CT31 cm6 molec 2 s 1 using argon as the bath gas at 300 K [53], As with the reaction of SO + 03 discussed above, collisional coupling results in a radiative lifetime that is pressure dependent. 

The lifetime of a homogeneous population of fluorophores is very often independent of the excitation wavelength as the emission spectrum (but there are some exceptions). In fact, internal conversion and vibrational relaxation are always very fast in solution and emission arises from the lowest vibrational level of state Si. 

Among other examples, time-resolved luminescence has recently been applied to the detection of different trace elements (i.e., elements in very low concentrations) in minerals. Figure 1.13 shows two time-resolved emission spectra of anhydrite (CaS04). The emission spectrum just after the excitation pulse (delay 0 ms) shows an emission band peaking at 385 nm, characteristic of Eu + ions. When the emission spectmm is taken 4 ms after the pulse, the Eu + luminescence has completely disappeared, as this luminescence has a lifetime of about 10/rs. This allows us to observe the weak emission signals of the Eu + and Sm + ions present in this mineral, which in short time intervals are masked by the En + Inminescence. The trivalent ions have larger lifetimes and their luminescence still remains in the ms delay range. 

The phosphine-functionalized tpy ligand 4 -Ph2Ptpy has both hard and soft donor sites the ligand is prone to oxidation to 4 -Ph2P(0)tpy and this affects the method of preparation of [Ru(4 -Ph2Ptpy)2]. " The complex [RuL(4,4 -Me2bpy)(NCS)] where L = 4-phosphonato-2,2 6, 2"-terpyridine, shows two ground-state pKi values at 6.0 and <4.0. The emission spectrum of the complex and the excited-state lifetimes are pFI-dependent. ... 

Bands in the emission spectrum—numbers to right show relative intensities. h The phosphorescence quantum yield is designated as the phosphorescence lifetime is designated... 

Weller24 has estimated enthalpies of exciplex formation from the energy separation vg, — i>5 ax of the molecular 0"-0 and exciplex fluorescence maximum using the appropriate form of Eq. (27) with ER assumed to have the value found for pyrene despite the doubtful validity of this approximation the values listed for AHa in Table VI are sufficiently low to permit exciplex dissociation during its radiative lifetime and the total emission spectrum of these systems may be expected to vary with temperature in the manner described above for one-component systems. This has recently been confirmed by Knibbe, Rehm, and Weller30 who obtain the enthalpies and entropies of photoassociation of the donor-acceptor pairs listed in Table XI. From a detailed analysis of the fluorescence decay curves for the perylene-diethyl-aniline system in benzene, Ware and Richter34 find that... 

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