Ultrafast electron-transfer and solvent adiabaticity effects in viologen charge-transfer complexes

We report ultrafast electron transfer (ET) in charge-transfer complexes that shows solvent relaxation effects consistent with adiabatic crossover models of nonadiabatic ET. The complexes of either dimethyl viologen (MV) or diheptyl viologen (HV) with 4,4'-biphenol (BP) (MVBP or HVBP complexes) have identical charge-transfer spectra and kinetics in ethylene glycol with approximately 900 fs ET decay. We assign this decay time as largely due to adiabatic control of a predicted approximately 40 fs nonadiabatic ET. The MVBP decay in methanol of 470 fs is reduced in mixtures having low (2-20%) concentrations of acetonitrile to as short as 330 fs; these effects are associated with faster relaxation time in methanol and its mixtures. In contrast, HVBP has much longer ET decay in methanol (730 fs) and mixture effects that only reduce its decay to 550 fs. We identify the heptyl substituent as creating major perturbations to solvent relaxation times in the methanol solvation shell of HVBP. These charge-transfer systems have reasonably well-defined geometry with weak electronic coupling where the electronic transitions are not dependent on intramolecular motions. We used a nonadiabatic ET model with several models for adiabatic crossover predictions to discuss the small variation of energy gap with solvent and the ET rates derived from adiabatic solvent control. A time correlation model of solvent relaxation was used to define the solvent relaxation times for this case of approximately zero-barrier ET.     

Fluorescence lifetimes and spectral properties of protochlorophyllide in organic solvents in relation to the respective parameters in vivo

In this work, absorption and fluorescence spectra of protochlorophyllide (Pchlide), as well as its fluorescence lifetime, were investigated in organic solvents having different physical properties. The obtained Pchlide spectral features are discussed in relation to the parameters describing solvent properties (refractive index and dielectric constant) and taking into account the specific solvent-Pchlide interaction. The correlation of Pchlide Qy and Soret absorption bands with solvent polarizability function ((n2 - 1)/(n2 + 2)) has been found; however, the dispersion of the observed points was rather high. A small Stokes shift of a magnitude between 50 and 300 cm(-1) was found, which indicates low sensitivity of Pchlide to nonspecific solvation. The fluorescence decay of Pchlide was single exponential in all the investigated solvents, with the lifetime value ranging from 5.2 ns for dioxane to 3.5 ns for methanol. Dependence of the obtained fluorescence lifetimes on the solvent orientation polarizability, a parameter being the function of both refractive index and dielectric constant, was discussed. In water-methanol mixtures, a further decrease of the fluorescence lifetime was observed, giving values of 2.9 ns for 25% methanol. Double-exponential decay of Pchlide fluorescence was found for Pchlide in a solution of 15% methanol with the lifetimes of 4.5 +/- 0.5 ns and 1.2 +/- 0.3 ns and in pure water with the lifetimes of 2.5 +/- 0.5 ns and 0.4 +/- 0.1 ns. The obtained results are discussed in relation to spectroscopic properties of Pchlide in vivo.     

Effect of the solvent composition in methanol-pentane and methanol-acetonitrile mixtures on the spectral and luminescent properties of 1,2-dihydroquinolines

The dependence of absorption and fluorescence spectra, quantum yields, and lifetimes of fluorescence on the solvent composition in the MeOH-C₅H₁₂ and MeOH-MeCN mixtures was studied for 2,2,4,6-tetramethyl-1,2-dihydroquinoline (TMDHQ). The variations in the parameters of deconvolution of the absorption and fluorescence spectra by the Gaussian functions in the MeOH-C₅H₁₂ mixtures of various compositions indicate the specificity of methanol clustering in saturated hydrocarbons and hydrogen bonding between TMDHQ and the methanol clusters of different compositions. At low MeOH concentrations (∼0.2 vol %), TMDHQ molecules are practically completely bound with the MeOH molecules by hydrogen bonds. In the MeOH-MeCN mixtures, the changes in the absorption and fluorescence spectra are observed at a substantially higher MeOH concentration (≥10 vol %) and monotonically change at the further increase in the MeOH concentration that is caused by the peculiarities of MeOH clustering in acetonitrile and the distribution of the TMDHQ molecules between the solvent components. At 50–95 vol % of MeOH in the mixture with MeCN, the fluorescence decay kinetics is described by the biexponential curve with the lifetime of the major component (τ₁) decreasing from 7.5 to 1.1 ns in pure MeCN and MeOH, respectively, and the lifetime of the minor component τ₂ ≈ 4 ns corresponding to the fluorescence lifetime in the solution containing 50 vol % MeOH. This indicates the existence of the free TMDHQ molecules, which are not bound with MeOH molecules or their clusters.     

苯乙烯Ziegler-Natta聚合扩散控制动力学研究

本文研究苯乙烯在TiCl_3-Al(C_2H_5)_3催化下于甲苯、正庚烷或甲苯-正庚烷混合物介质中的聚合动力学。提出了一个含催化剂球形聚合物颗粒中发生单体扩散控制聚合的模型,导出的式子统一解释了不同介质中扩散阻力造成的不同程度的速率表减,定量说明了在聚笨乙烯良溶剂中聚合时扩散阻力小于在不良溶剂中时。     

Extraction of lifetime distributions from fluorescence decays with application to DNA-base analogues

Several important aspects of fluorescence decay analysis are addressed and tested against new experimental measurements. A simulated-annealing method is described for deconvoluting the instrument response function from a measured fluorescence decay to yield the true decay, which is more convenient for subsequent fitting. The method is shown to perform well against the conventional approach, which is to fit a convoluted fitting function to the experimentally measured decay. The simulated annealing approach is also successfully applied to the determination of an instrument response function using a known true fluorescence decay (for rhodamine 6G). The analysis of true fluorescence decays is considered critically, focusing specifically on how a distribution of decay constants can be incorporated in to a fit. Various fitting functions are applied to the true fluorescence decays of 2-aminopurine in water-dioxane mixtures, in a dinucleotide, and in DNA duplexes. It is shown how a suitable combination of exponential decays and non-exponential decays (based on a Γ distribution of decay constants) can provide fits of equal quality to the conventional multi-exponential fits used in the majority of previous studies, but with fewer fitting parameters. Crucially, the new approach yields decay-constant distributions that are physically more meaningful than those corresponding to the conventional multi-exponential fit. The methods presented here should find wider application, for example to the analysis of transient-current or optical decays and in F?rster resonance energy transfer (FRET).     

Photophysical properties of β-(1-pyrenyl)ethyl p-cyanobenzoate in binary solvents of isooctane-ethyl acetate and ethyl acetate-acetonitrile

The effects of solvent polarity on the fluorescence spectra and fluorescence decays of β-(1-pyrenyl)ethyl p-cyanobenzoate (P2CN) were investigated in detail using binary solvents consisting of various mixing ratios of isooctane-ethyl acetate or ethyl acetate-acetonitrile (dielectric constants ()=1.94–36.2). Whereas both the intensity and wavelength maxima of an intramolecular exciplex emission (EX) are dependent on the solvent polarity, only the intensity of an emission from the locally excited pyrene (LE) is dependent on the solvents used. When monitored at 377 nm, the picosecond SPC (single photon counting) measurements reveal a slow decay (>150 ns) in addition to a fast decay (<1 ns) of the locally excited P2CN. There are also two decays for the EX which vary the intensity ratios by the monitored wavelength. The decay rate constants, k_EX1 and k_EX2, have a good linear correlation with the dielectric constants of the solvents, indicating that there exist two kinds of exciplexes. It is suggested that the decays of the locally excited-state of P2CN are so fast due to result of the efficient electron transfer that the two kinds of intramolecular exciplexes are formed from the two discrete conformers in the ground state.     

Nanosecond pulse radiolysis studies in benzene-methanol mixtures using a rigid 7-amino 4-trifluoro methyl coumarin as a probe

Pulse radiolysis of benzene-methanol mixtures leads to the formation of triplets and dimer anions of the solute C153. The spectra of the dimer anions and triplet in these solvent mixtures are reported. The relative G-values of these transients are determined. Dimer anion yield was found to vary exponentially with the reciprocal of the dielectric constant of the mixture.     

Time-resolved emission of flavin adenine dinucleotide in water and water-methanol mixtures

Time-resolved fluorescence decay of flavin adenine dinucleotide (FAD) was studied at room temperature in water and water-methanol mixtures by a fluorescence upconversion technique. The observations were focused on the most initial decay phase (200 ps), before the residual fluorescence assumes a single exponential decay, typical for an extended conformation of the fluorophore. Within the first few picoseconds, where most of the electron transfer coupled quenching takes place, the emission decay curves could be fitted by a stretched exponent, compatible with the inhomogeneous distance dependent electron transfer model. This implies that the population of the excited FAD molecules exhibits a large number of non-identical states, each with its own separation between the donor (adenine) and acceptor (isoalloxazine) moieties, having its own rate of electron transfer. To evaluate the distribution of the separation between the donor-acceptor pair, we carried out molecular dynamics simulations of closed conformation of the FAD in water and water-methanol mixtures, sampling the structure at 10 fs intervals. The analysis of the dynamics reveals that within the 4 ps time frame, where most of the nonexponential fluorescence relaxation takes place, the relative motion of the donor-acceptor pair is consistent with a one-dimensional Brownian motion, where the diffusion coefficient and the shape of the confining potential well are solvent dependent. The presence of methanol enhances the diffusion constant and widens the width of the potential well. On the basis of these parameters, the relaxation dynamics was accurately reconstructed as an electron transfer reaction in an inhomogeneous system where the reactants are diffusing within the time frame of the observation.     

Fluorescence decay of α-chymotrypsin studied by the picosecond-resolved single photon-counting technique

The fluorescence decay of the multi-tryptophan-containing enzyme α-chymotrypsin in Tris buffer (pH 7.8) at room temperature was studied using a frequency-doubled, synchronously-pumped picosecond rhodamine-6G laser excitation source with time-correlated single photon-counting detection. The fluorescence decay parameters were computed with a non-linear least-squares iterative reconvolution program. The goodness-of-fit was tested with well-known graphical methods such as residuals plots and the autocorrealtion function. Numerical tests (reduced chi-square, ordinary runs test and the Durbin—Watson statistic) were included to improve the reliability of the residuals analysis. Normal distribution of the weighted residuals was checked with the normal probability plot, and with computation of the mean and standard deviation of the weighted residuals. α-Chymotrypsin exhibited triple-exponential fluorescence decay kinetics with decay times of 615 ± 76 ps, 1.7 ± 0.2 ns, and 4.3 ± 0.3 ns. The fractional fluorescence contributions depended on the emission wavelength. The fluorescence spectra of the components contributing to the total fluorescence were calculated from the steady-state fluorescence spectrum and fluorescence decays at different emission wavelengths, and from convoluted time-resolved emission spectra and a fluorescence decay measurement.     

Time-resolved fluorescence reveals two binding sites of 1,8-ANS in intact human oxyhemoglobin

Time-resolved fluorescence of 1,8-anilinonaphthalene sulfonate (1,8-ANS) fluorescent probe bound to intact human oxyhemoglobin (HbO2) is investigated. Fluorescence emission spectra of 1,8-ANS in a potassium buffer solution (pH 7.4) of HbO2 undergo a substantial blue shift during first 6 ns after pulsed optical excitation at 337.1 nm. Nonexponential fluorescence kinetics of 1,8-ANS in the HbO2 solution are studied by the decay time distribution and conventional multiexponential analyses for a set of emission wavelength range of lambdaem = 455-600 nm. These fluorescence decays contain components with mean decay times of <0.5 ns, 3.1-5.5 ns, and 12.4-15.1 ns with spectrally-dependent relative contributions. The shortest decay component is assigned to free 1,8-ANS molecules in the bulk buffer environment, whereas the two longer decay components are assigned to two types of binding sites of 1,8-ANS in the HbO2 molecule presumably differing by polarity and accessibility to water molecules. The results represent the first experimental evidence of heterogeneous binding of 1,8-ANS to intact human oxyhemoglobin.     

Solvent reorganization controls the rate of proton transfer from neat alcohol solvents to singlet diphenylcarbene

Femtosecond transient absorption spectroscopy was used to study singlet diphenylcarbene generated by photodissociation of diphenyldiazomethane with a UV pulse at 266 nm. Absorption by singlet diphenylcarbene was detected and characterized for the first time. Similar band shapes were observed in acetonitrile and in cyclohexane with lambda(max) approximately 370 nm. The singlet absorption decays by intersystem crossing to triplet diphenylcarbene at rates that agree with previous measurements. The singlet absorption band is completely formed 1 ps after the pump pulse. It is preceded by a strong and broad absorption band, which is tentatively assigned to excited-state absorption by a singlet diazo excited state. In neat alcohol solvents the growth and decay of the diphenylmethyl cation was observed. This species is formed by proton transfer from an alcohol molecule to singlet diphenylcarbene. Since a shell of solvent molecules surrounds each nascent carbene, the intrinsic rate of protonation in the absence of diffusion could be measured. In methanol, proton transfer occurs with a time constant of 9.0 ps, making this the fastest known intermolecular proton-transfer reaction to carbon. In O-deuterated methanol proton transfer occurs in 15.0 ps. Slower rates were observed in the longer alcohols. The protonation times correlate reasonably well with solvation times in these alcohols, suggesting that solvent fluctuations are the rate-limiting step. In all alcohols studied, the carbocations decay on a somewhat slower time scale to yield diphenylalkyl ethers. In methanol and ethanol the rate of decay is determined by reaction with neutral solvent nucleophiles. There is evidence in 2-propanol that geminate reaction within the initial ion pair is faster than reaction with solvent. No isotope effect was observed for the reaction of the diphenylmethyl carbocation in methanol. Using comparative actinometry the quantum yield of protonation was measured. In methanol, the quantum yield of carbocations reaches a maximum value of 0.18 approximately 18 ps after the pump pulse. According to our analysis, 30% of the photoexcited diazo precursor molecules are eventually protonated. Somewhat lower protonation efficiencies are observed in the other alcohols. Because the primary quantum yield for formation of singlet diphenylcarbene is still unknown, the importance of reaction channels that might exist in addition to protonation cannot be determined at present. Singlet carbenes are powerful, photogenerated bases that open new possibilities for fundamental studies of proton transfer in solution.     

Intriguing H-aggregate and H-dimer formation of coumarin-481 dye in aqueous solution as evidenced from photophysical studies

Photophysical properties of coumarin-481 (C481) dye in aqueous solution show intriguing presence of multiple emitting species. Concentration and wavelength dependent fluorescence decays and time-resolved emission spectra and area-normalized emission spectra suggest the coexistence of dye monomers, dimers, and higher aggregates (mostly trimers) in the solution. Because of the efficient intramolecular charge transfer (ICT) state to twisted intramolecular charge transfer (TICT) state conversion, the dye monomers show very short fluorescence lifetime of ~0.2 ns. Fluorescence lifetimes of dimers (~4.1 ns) and higher aggregates (~1.4 ns) are relatively longer due to steric constrain toward ICT to TICT conversion. Observed results indicate that the emission spectra of the aggregates are substantially blue-shifted compared to monomers, suggesting H-aggregation of the dye in the solution. Temperature-dependent fluorescence decays in water and time-resolved fluorescence results in water-acetonitrile solvent mixtures are also in support of the dye aggregation in the solution. Though dynamic light scattering studies could not recognize the dye aggregates in the solution due to their small size and low concentration, fluorescence up-conversion measurements show a relatively higher decay tail in water than in water-acetonitrile solvent mixture, in agreement with higher dye aggregation in aqueous solution. Time-resolved fluorescence results with structurally related non-TICT dyes, especially those of coumarin-153 dye, are also in accordance with the aggregation behavior of these dyes in aqueous solution. To the best of our knowledge, this is the first report on the aggregation of coumarin dyes in aqueous solution. Present results are important because coumarin dyes are widely used as fluorescent probes in various microheterogeneous systems where water is always a solvent component, and the dye aggregation in these systems, if overlooked, can easily lead to a misinterpretation of the observed results.     

Self-assembly of a hydrophobically modified naphthalene-labeled poly(acrylic acid) polyelectrolyte in water: organic solvent mixtures followed by steady-state and time-resolved fluorescence

The solution properties of two water-soluble polymers, poly(acrylic acid) (PAA), covalently labeled with the fluorescent hydrophobic dye naphthalene (Np), have been investigated in water:organic solvent mixtures. The naphthalene chromophores have been randomly attached, onto the polymer, with two different degrees of labeling. Fluorescence measurements (steady-state and time-resolved) have been used to follow the photophysical behavior of the polymers and consequently report on the self-association of the polymers in the mixed organic (methanol or dioxane):aqueous solutions. The emission spectra of the high-labeled Np PAA reveal the presence of monomer and excimer bands whereas with the low-labeled polymer only monomer emission is observed. The excitation spectra collected at the monomer and excimer emission bands show significant differences, depending on the water content of the mixture, which indicate the simultaneous presence of preformed and dynamic dimers as routes to excimer formation. The time-resolved data decay profiles of the high-labeled polymer in the mixtures were always triple exponential whereas in pure methanol and dioxane they follow biexponential laws. The data in the mixtures are consistent with two types of monomers and one excimer. Both monomers are able to give rise to excimer in the excited state, one type involving the movement of long distant Np chromophores and the other involving a local reorientation of adjacent Np chromophores. These correspond to different decay times: (1) a long which corresponds to the long distant approach of non-neighboring Np chromophores forming an excimer and (2) a short corresponding to the fast adjustment of two neighboring Np chromophores in order to have the adequate parallel geometry. An additional decay time corresponding to the excimer decay was found to be present at all wavelengths. All the decay times were dependent on the water content of the mixture. An estimation of the two excimer forming rate constants values is made for the mixed media considered in this work. On the whole, using both steady-state and time-resolved fluorescence parameters, and by comparing data for a polymer with a small number of hydrophobes with a more highly modified one, it is possible in great detail to demonstrate how association is controlled by solvent quality for the hydrophobes and by the distance between hydrophobes.     

Rotational reorientation dynamics of oxazine 750 in polar solvents

The rotational reorientation dynamics of oxazine 750 (OX750) in the first (with pump pulse at 660 nm) and a higher excited state (with pump pulse at 400 nm) in different polar solvents have been investigated using femtosecond time-resolved stimulated emission pumping fluorescence depletion (FS TR SEP FD) spectroscopy. In both excited states, three different anisotropy decay laws have been observed for OX750 in different solvents. Only in acetone and formamide could the anisotropy decays of OX750 be described by single-exponential functions, whereas the anisotropy decays have been found to exhibit biexponential behavior in other solvents. The slower anisotropy decay observed in all of the solvents has been assigned to the overall rotational relaxation of OX750 molecules, and a quantitative analysis of this time constant has been performed using the Stokes-Einstein-Debye hydrodynamic theory and the extended charge distribution model developed by Alavi and Waldeck. In both methanol and ethanol, a faster anisotropy decay on the order of picoseconds and a slower anisotropy decay on the hundreds of picoseconds time scale are observed. The most likely explanation for the faster anisotropy involves the rotation of the transition dipole moment in the excited state of OX750 resulting from the electron transfer (ET) reaction taking place from the alcoholic solvents to the OX750 chromophore. As a possible explanation, the wobbling-in-the-cone model has been used to analyze the biexponential anisotropy decays of OX750 in dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). The observed faster anisotropy decays on the hundreds of femtoseconds time scale in DMF and DMSO are ascribed to the wobbling-in-the-cone motion of the ethyl group of OX750, which is sensitive to the strength of the hydrogen bond formed between the solvent and the protonation site of OX750.     

Dissociation constants of phenols in methanol--water mixtures

A preferential solvation model that relates solute properties with solvent composition in binary mixtures has been applied to the dissociation pKa values of a set of 28 substituted phenols in methanol-water mixtures. The parameters of the model allow estimation of the pKa value of each phenol for any methanol-water composition. Moreover, it is demonstrated that the pKa values of the whole set of phenols at any methanol-water composition are linearly related to the pKa values of the phenols in water. Equations that relate the correlations' slope and intercept values with the solvent composition have been derived and tested with the set of phenols. The general parameters obtained for these equations allow an accurate calculation of the pKa value of any phenol, even of those not included in the original set, at any methanol-water composition solely from the pKa value of the phenol in water. These calculated pKa values can be used for quantitative structure-HPLC retention relationships. The method is tested by comparison of the calculated pKa values with the HPLC determined pKa values of 26 phenols in a polymeric column with a 50% methanol as mobile phase.     

Molecular dynamics simulations of porphyrin-dendrimer systems: toward modeling electron transfer in solution

We have performed computational simulations of porphyrin-dendrimer systems--a cationic porphyrin electrostatically associated to a negatively charged dendrimer--using the method of classical molecular dynamics (MD) with an atomistic force field. Previous experimental studies have shown a strong quenching effect of the porphyrin fluorescence that was assigned to electron transfer (ET) from the dendrimer's tertiary amines (Paulo, P. M. R.; Costa, S. M. B. J. Phys. Chem. B 2005, 109, 13928). In the present contribution, we evaluate computationally the role of the porphyrin-dendrimer conformation in the development of a statistical distribution of ET rates through its dependence on the donor-acceptor distance. We started from simulations without explicit solvent to obtain trajectories of the donor-acceptor distance and the respective time-averaged distributions for two dendrimer sizes and different initial configurations of the porphyrin-dendrimer pair. By introducing explicit solvent (water) in our simulations, we were able to estimate the reorganization energy of the medium for the systems with the dendrimer of smaller size. The values obtained are in the range 0.6-1.5 eV and show a linear dependence with the inverse of the donor-acceptor distance, which can be explained by a two-phase dielectric continuum model taking into account the medium heterogeneity provided by the dendrimer organic core. Dielectric relaxation accompanying ET was evaluated from the simulations with explicit solvent showing fast decay times of some tens of femtoseconds and slow decay times in the range of hundreds of femtoseconds to a few picoseconds. The variations of the slow relaxation times reflect the heterogeneity of the dendrimer donor sites which add to the complexity of ET kinetics as inferred from the experimental fluorescence decays.     

Analyzing nonexponential kinetics with multiple population-period transient spectroscopy (MUPPETS)

A new multidimensional spectroscopy (MUPPETS) was recently introduced (van Veldhoven, E.; et al. ChemPhysChem 2007, 8, 1761) that distinguishes between nonexponential relaxations that are due to heterogeneous dynamics and those that are due to homogeneous dynamics. This paper develops methods for the quantitative analysis of MUPPETS data and demonstrates the ability of this experiment to decompose a complex decay into its components. These methods have been applied to MUPPETS data on the ground-state recovery of auramine in methanol and on a mixture of auramine and coumarin 102 in methanol. The auramine is found to have two kinetically different components, even though the decay times are too similar to be distinguished in a one-dimensional experiment. The dynamics of each component are derived from the MUPPETS data in a model-free procedure in particular without assuming that the individual decays are exponential or that they have similar shapes. In fact, the component decays are each found to be nonexponential and to have different decay shapes. We suggest that the two components are due to ion-paired and nonpaired molecules. The effect of rotation on MUPPETS with all parallel polarizations is analyzed. The nonexponentiality in ground-state recovery signals due to the combination of rotation and population decay is shown to behave as a nearly ideal homogeneous nonexponentiality. This prediction is confirmed in a mixture of auramine and coumarin. MUPPETS allows the decay from the fast relaxing auramine to be removed from the mixture, leaving only the rotation/population decay of the coumarin.     

Foldamer dynamics expressed via Markov state models. I. Explicit solvent molecular-dynamics simulations in acetonitrile, chloroform, methanol, and water

In this article, we analyze the folding dynamics of an all-atom model of a polyphenylacetylene (pPA) 12-mer in explicit solvent for four common organic and aqueous solvents: acetonitrile, chloroform, methanol, and water. The solvent quality has a dramatic effect on the time scales in which pPA 12-mers fold. Acetonitrile was found to manifest ideal folding conditions as suggested by optimal folding times on the order of approximately 100-200 ns, depending on temperature. In contrast, chloroform and water were observed to hinder the folding of the pPA 12-mer due to extreme solvation conditions relative to acetonitrile; chloroform denatures the oligomer, whereas water promotes aggregation and traps. The pPA 12-mer in a pure methanol solution folded in approximately 400 ns at 300 K, compared relative to the experimental 12-mer folding time of approximately 160 ns measured in a 1:1 v/v THF/methanol solution. Requisite in drawing the aforementioned conclusions, analysis techniques based on Markov state models are applied to multiple short independent trajectories to extrapolate the long-time scale dynamics of the 12-mer in each respective solvent. We review the theory of Markov chains and derive a method to impose detailed balance on a transition-probability matrix computed from simulation data.     

Analysis of Fluorescence Lifetime of Protochlorophyllide and Chlorophyllide in Isolated Etioplast Membranes Measured from Multifrequency Cross-correlation Phase Fluorometry

The fluorescence decays of protochlorophyllide (Pchlide) and of chlorophyllide (Chlide) in wheat etioplast membranes were analyzed using a multiexponential fluorescence decay model. Using different excitation wavelengths from 430 to 470 nm, we found that a triple-exponential model at 14°C and a double-exponential model at — 170°C were adequate to describe the Pchlide fluorescence decay. We discuss the origin of the three fluorescence lifetime components at 14°C on the basis of the dependence of their fractional intensities on the excitation wavelength and by correlating the fractional intensities with integrated fluorescence intensities of different Pchlide forms in steady-state fluorescence spectra. The fluorescence decay of the main Pchlide form, photoactive Pchlide-F657, is shown to have a complex character with a fast component of 0.25 ns and a slower component of about 2 ns. Two lifetime components of 2 ns and 5.5–6.0 ns are ascribed to the second photoactive form, Pchlide-F645, and to nonphotoactive Pchlide forms, respectively. In etioplast membranes preilluminated by a short saturating light pulse, we found a single 5.0 ns component for Chlide-F688 (the Chlide-NADPH: protochlorophyllide oxidoreductase [PORJ-NADP⁺complex) and an additional 1.6 ns component when the formation of Chlide-F696 (the Chlide-POR-NADPH complex) was promoted by exogenous NADPH. From the fluorescence lifetime results we evaluated the quantum yield of the primary photoreaction by Chlide-F696 as being 70%.     

RESOLUTION OF TWO EMISSION SPECTRA FOR TRYPTOPHAN USING FREQUENCY-DOMAIN PHASE-MODULATION SPECTRA

We describe a novel application of frequency-domain fluorometry which allows resolution of the decay times and emission spectra of samples which display multi-exponential decay kinetics. This method does not require any previous knowledge about the decay times or any assumptions about the shape of the emission spectra. We record the wavelength-dependent phase angles and modulations (phase angle and modulation spectra) using a number of light modulation frequencies. The data is analyzed by non-linear least-squares to recover the emission spectra and their associated decay times. Phase and modulation spectra (PM Spec) were used to recover the emission spectra associated with the two decay times of tryptophan at pH = 7 (0.54 and 3.44 ns). The emission spectra of these components are centered at 340 and 355 nm, respectively, with the amplitude of the 0.54 ns component contributing 6% to the total emission. These results are in agreement with previous time-resolved studies by Szabo and Rayner [J. Am. Chem. Soc. 102, 554-563 (1980)]. Control experiments were performed on mixtures of N-acetyl-L-tryptophanamide (NATA) and PPD, which demonstrate our ability to recover the spectra and decay times from two component mixtures. NATA itself displayed a single decay time and only one emission spectrum.