Energy transfer experiments

On the experimental side, small molecule energy transfer experiments may use molecular beam teclmiques [65. 66 and 67] (see also chapter C3.3 for laser studies). 

As a first step in imderstanding the analysis of energy transfer experiments, it is wortliwhile to summarize tire steps in a typical experiment where CgFg is tire hot donor and carbon dioxide is tire bath receptor molecule. First, excited... 

Fluorescence energy transfer experiments, in which the energy transfer from the excited DNA bases to a fluorescent ligand is monitored by fluorescence excitation spectroscopy, has been used to analyze the binding of the bisquinolizinium species 35 to DNA <2004ARK219>. 

Energy-transfer experiments involving sensitisation and quenching allow us to selectively populate and depopulate excited states. This affords us a powerful tool whereby the particular excited state... 

The above examples show that a very important criterion in the choice of a probe is its sensitivity to a particular property of the microenvironment in which it is located (e.g. polarity, acidity, etc.). On the other hand, insensitivity to the chemical nature of the environment is preferable in some cases (e.g. in fluorescence polarization or energy transfer experiments). Environment-insensitive probes are also better suited to fluorescence microscopy and flow cytometry. 

The definition used depends on the phenomenon under study. For instance, the intensity-averaged lifetime must be used for the calculation of an average colli-sional quenching constant, whereas in resonance energy transfer experiments, the amplitude-averaged decay time or lifetime must be used for the calculation of energy transfer efficiency (see Section 9.2.1). 

A schematic diagram of the apparatus used in the energy transfer experiments is shown in Figure 8.22. The particles are produced and levitated in an electrodynamic levitator as described previously. Excitation is provided by the filtered output of either a Xe or Hg-Xe high-pressure arc. The intensity produced at the particle was found to be 10-50 mW/cm2. The fluorescence emitted from each of the levitated particles was monitored at 90° to the exciting beam using //3 optics, dispersed with a j-m monochromator, and detected with an optical multichannel analyzer. The levitator could be... 

Figure 8.22. Schematic diagram of the apparatus used in the energy transfer experiments.

Details of nitrobenzene photochemistry reported by Testa are consistent with the proposal that the lowest triplet excited state is the reactive species. Photoreduction, as measured by disappearance quantum yields of nitrobenzene in 2-propanol is not very efficient = (1.14 0.08) 10 2 iD. On the other hand, the triplet yield of nitro benzene in benzene, as determined by the triplet-counting method of Lamola and Hammond 28) is 0.67 0.10 2). This raises the question of the cause of inefficiency in photoreduction. Whereas Lewis and Kasha 29) report the observation of nitrobenzene phosphorescence, no long-lived emission from carefully purified nitrobenzene could be detected by other authors i4,3o). Unfortunately, the hterature value of Et for nitrobenzene (60 kcal mole i) is thus based on an impurity emission and at best a value between 60 and 66 kcal mole can be envisaged from energy-transfer experiments... 

A short lifetime (t = 10 s) for the lowest excited triplet of nitrobenzene has also been obtained from energy-transfer experiments using cis-piperylene as quencher. Even at concentrations of 2.4 mole 1 cts-piperylene not all nitrobenzene triplets were quenched but singlet energy-transfer has been disfavored by kinetic reasoning... 

Energy transfer experiments permit the measurement of distances because the rate of transfer is proportional to the inverse sixth power of the distance between donor and acceptor. According to the theory of Forster the donor-acceptor distance r is related to the transfer efficiency E by eq 16... 

The possibility to carry out conformational studies of peptides at low concentrations and in the presence of complex biological systems represents a major advantage of fluorescence spectroscopy over other techniques. Fluorescence quantum yield or lifetime determinations, anisotropy measurements and singlet-singlet resonance energy transfer experiments can be used to study the interaction of peptides with lipid micelles, membranes, proteins, or receptors. These fluorescence techniques can be used to determine binding parameters and to elucidate conformational aspects of the interaction of the peptide with a particular macro-molecular system. The limited scope of this chapter does not permit a comprehensive review of the numerous studies of this kind that have been carried and only a few general aspects are briefly discussed here. Fluorescence studies of peptide interactions with macromolecular systems published prior to 1984 have been reviewed. 

The energy of the relaxed S state of alkanes in the liquid phase is certainly somewhere between the energy of the absorption onset and the energy of the fluorescence maximum. In energy-transfer experiments, the energy of absorption threshold is usually... 

Based on energy-transfer experiments in some works, low-energy (and possibly long-living) alkane triplet state is also indicated however, there is no direct proof for its existence [7,36,37]. 

K = k T. When energy-transfer experiments were used to deduce the x values, the quenching was assumed to proceed with a diffusion-controlled rate, and the transfer was supposed to take place when the acceptor and donor molecules were in contact (no static quenching). The transfer rate coefficients were calculated using the usual diffusional equations [24,25,59] (see Sec. 3.1). 

The yield determined in a certain type of experiment usually strongly depends on the assumptions made about the formation mechanism. In the older literature, the excited molecules were often assumed to be produced solely in neutral excitations [127,139-143] and energy-transfer experiments with Stern-Volmer-type extrapolation (linear concentration dependence) were used to derive G(5 i). For instance, by sensitization of benzene fiuorescence, Baxendale and Mayer established G(5 i) = 0.3 for cyclohexane [141]. Later Busi [140] corrected this value to G(5 i) = 0.51 on the basis that in the transfer, in addition to the fiuorescing benzene state S, the S2 and S3 states also form and the 82- 81 and 83 81 conversion efficiencies are smaller than 1. Johnson and Lipsky [144] reported an efficiency factor of 0.26 0.02 per encounter for sensitization of benzene fluorescence via energy transfer from cyclohexane. Using this efficiency factor the corrected yield is G(5 i) = 1.15. Based on energy-transfer measurements Beck and Thomas estimated G(5 i) = 1 for cyclohexane [145]. Relatively small G(5 i) values were determined in energy-transfer experiments for some other alkanes as well -hexane 1.4, -heptane 1.1 [140], cyclopentane 0.07 [142] and 0.12 [140], cyclooctane 0.07 [142] and 1.46 [140], methylcyclohexane 0.95, cifi-decalin 0.26 [140], and cis/trans-decalin mixture 0.15 [142]. 

A basic problem of the energy-transfer experiments is that the quenchers used may also react with electron or cationic species, in addition to the excited molecules. If the excited molecules also form after charge recombination (which is now unambiguously established), the quenchers may considerably hinder the formation of excited molecules [8]. Comparison of the results obtained in this manner with those obtained via other techniques shows that the solute technique strongly underestimates the G(Si) value. It should be mentioned that in most of the sensitization experiments unrealistically high K Stern-Volmer constants were obtained [141,142]. 

Figure 13.22. Singlet-triplet gap in stable nitrenium ion probed by energy transfer experiments.

Fig. 16. Structures of the tetracationic porphyrin (TAPP) donor and the tricationic cyanine dye (Cy3+) acceptor used in the polymerization-enhanced energy transfer experiments.

SAOTS was established to be a true monolayer (rather than a bi- or multilayer) by conductivity and Forster-type energy transfer measurements [183]. Energy transfer experiments were carried out on a composite system which consisted of a mixed OTS and donor cyanine dye (D) monolayer on a glass slide, on top of which a mixed cadmium arachidate acceptor cyanine dye (A) monolayer was deposited (by the LB technique) in such a manner that it covered only half of the glass slide. The other half was covered by a pure... 

SINGLE MODE DYE LASER BEAM Figure 9. Schematic of energy-transfer experiment. 

Again this implies that no distinction is made between differing energy levels above the critical. Energy transfer experiments show that this is a better approximation than the corresponding activation assumption, in so far as one collision is often... 

Green fluorescent protein is commonly used for energy-transfer experiments (Baubet etal. 2000). The fluorescent moiety of GFP protein is the Ser-Tyr-Gly derived chromophore. GFP can be expressed in a variety of cells where it becomes fluorescent, can be fused to a host... 

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