MECHANISM OF PHOTOREDUCTION OF THIAZINE DYES BY EDTA STUDIED BY FLASH PHOTOLYSIS-II. pH DEPENDENCE OF ELECTRON ABSTRACTION RATE CONSTANT OF THE DYES IN THEIR TRIPLET STATE

Abstract— The pH dependence of the apparent reactivity of thiazine dyes in their triplet states has been studied in aqueous solutions, using as electron donor HY^-3, the trianionic species of ethylene diamine tetraacetic acid (EDTA), in the pH range 4–8. The pH dependence is found to be related to a change in the degree of protonation of the triplet excited dye. The apparent reactivity and lifetime of two differently protonated forms of thionine, azur B and methylene blue were determined by classical and dye-laser flash techniques, making it possible to evaluate the rate constant for electron abstraction of these molecules in their triplet states. It is found that: (a) protonation on the ring nitrogen increases the electron-abstraction rate constant of the triplet-state species about twenty-fold, and (b) methylation on the side amino groups decreases it.     

PHOTOCHEMISTRY OF FLAVINS. II. PHOTOPHYSICAL PROPERTIES OF ALLOXAZINES AND ISOALLOXAZINES

Abstract. Pulsed laser photolysis at 347nm has been used to study the transient spectroscopy of alloxazine, lumichrome, lumiflavin, and riboflavin in acidic (pH 2.2) aqueous solution and in ethanol. Intersystem crossing quantum yields (φ_ISC) were determined by a modification of the comparative laser excitation method which utilizes the variation of the triplet yield with intensity in conjunction with a kinetic model for the various photophysical and photochemical processes occurring during the pulse. Fluorescence quantum yields and lifetimes are also reported. Correction for quenching of the excited singlet state by H⁺ ions shows that, in neutral aqueous solution, intersystem crossing for flavins is an efficient process (φ_ISC˜ 0.7) which, in conjunction with fluorescence, accounts for the fate of all absorbed photons. For alloxazine (φ_ISC˜ 0.45) and lumichrome (φ_ISC˜ 0.7) the results are more difficult to interpret owing to interconversion between alloxazine and isoalloxazine structures in the singlet excited state. For all four compounds, the quantum yield of products derived from the singlet excited state is estimated as ˜0.04. There is evidence of biphotonic product formation at high laser energies. In ethanol, where φ_ISC for lumichrome is about twice that of lumiflavin, internal conversion between the excited singlet and ground states appears to be a significant process. Complete triplet-triplet absorption spectra in the region 260–750nm are reported. For lumichrome at pH 2.2 there is spectral evidence for isomeric triplet states which appear to be in equilibrium.     

ÉTUDE EN SPECTROSCOPIE PAR ÉCLAIR DES COLORANTS THIAZINIQUES EN SOLUTION AQUEUSE

Abstract— p K values of ionisation equilibrium of thiazines dyes in their triplet state have been measured in aqueous solutions by spectroscopy. The triplet-triplet absorption bands, in the red part of the spectrum, are given for thionine, azurs and methylene blue. It is shown that, in the pH range 4–9, the equilibria investigated correspond to a second protonation of thiazines dyes that occurs in the triplet state:
³TH⁺+ H⁺→³TH₂²⁺
designating by ³TH⁺ the thiazine cation similar to the species stable in neutral aqueous solutions.     

CHLORIDE ENHANCEMENT OF QUENCHING OF TRIPLET METHYLENE BLUE BY Fe(II)

Abstract— The influence of chloride ion on the rate of decay of triplet methylene blue in 0.01 M acid in the absence and presence of ferrous ions was investigated by means of laser flash-photolysis monitored by kinetic spectrophotometry. Chloride weakly accelerates decay of ³MBH^2± in aqueous solution in the absence of Fe(II). Quenching of ³MBH²⁺ by Fe(II) is more strongly catalyzed by Cl^- in both water and 50 v/v% aq. CH₃CN. The uncatalyzed quenching constant, k ₅, is of the order of 1 × 10⁶ M ^-1 s^-1 while in 4.8 M aqueous chloride ( μ – 7.2 M ) k ₅= (37.2 ± 1.8) × 10⁶ M ^-1 s^-1. A possible role of chloride is as a bridging species in quenching via electron transfer between ³MBH²⁺ and Fe(II).     

EFFECTS OF SOLVENT ON THE FLUORESCENCE PROPERTIES OF BACTERIOCHLOROPHYLL a

Fluorescence lifetimes (τ_f) of bacteriochlorophyll a (BChl a ) have been measured by the method of time-correlated single-photon counting on dilute (1 μ M ) solutions of the pigment in 15 solvents. There is a pronounced dependence of τ_f on the nature of the solvent. Specifically, τ_f, is longer when the central magnesium is hexacoordinated than when pentacoordinated and shorter when the macrocycle is hydrogen-bonded than when it is not, but the latter effect is more pronounced. Both trends were confirmed by parallel studies on bacteriopheophytin a (BPheo a ). Because of the short lifetimes (˜ 2.2–3.6 ns), quenching of fluorescence by molecular oxygen is not a significant factor in aerated solutions of the bacterial pigments. However, reabsorption artifacts are non-negligible, which necessitates studies on dilute solutions. Fluorescence quantum yields (ø_f) have been estimated for BChl a in 13 solvents by comparing the observed fluorescence lifetimes with the radiative lifetimes calculated from the integrated absorption spectra.     

FLUORESCENCE LIFETIMES OF CHLOROPHYLL a: SOLVENT, CONCENTRATION AND OXYGEN DEPENDENCE

Fluorescence lifetimes (τ_f) of chlorophyll a (Chi a ) have been measured by the single-photon-counting technique over a wide range of concentrations (˜10^-7˜10^-4 M ) in deoxygenated pyridine, diethyl ether, toluene and methanol. At pigment concentrations ˜1 μ M , reabsorption of fluorescence induces significant artifacts on measured values of τ_f which are dependent on detection wavelength and the specific geometry of the experiment. There is a clear dependence of τ_f on the nature and degree of solvation, including both coordination of the central magnesium and hydrogen-bonding of the solvent (viz. alcohols) to the macrocycle. Quenching of the excited singlet state by molecular oxygen was measured quantitatively in ether, and a bimolecular rate constant markedly slower than the diffusion-controlled limit was obtained.     

INCREASED QUANTUM YIELD OF PHOTOREDUCTION OF DYES (A CATALYTIC EFFECT)

Abstract— While studying the photoreduction of some dyes (D) by reducing agents (R), it was observed that the quantum yield of the photoreduction increases considerably upon addition of a third substance (C), whereas it is very small when the dye is photoreduced by C alone (catalytic effect), (see Table 1).
The system thionine (D), allylthiourea (R), and azulene (C) was investigated in detail using both flash photolysis and continuous illumination. On photolysis, thionine is converted into its photo-reduced form, leucothionine. Azulene reacts with the basic form of the thionine triplet ³ TH ⁺ to produce the semithionine radical. In the system thionine and azulene, most of these radicals revert back to thionine. When ATU (˜ 10^2- M ) is added to thionine and azulene (3 × 10^-4 M ), the semithionine radicals are reduced to leucothionine; the quantum yield of this reduction is considerably higher than in the system thionine and allylthiourea. Flash experiments demonstrate that allylthiourea does not react with the semithionine radicals.
At very high ATU concentrations (≥ 10^-1 M ), however, the primary reaction is between thionine triplet and allylthiourea; under these conditions the quantum yield is not influenced by azulene (3 × 10^-4 M ).     

MECHANISM OF PHOTOREDUCTION OF THIAZINE DYES BY EDTA STUDIED BY FLASH PHOTOLYSIS—III CONSEQUENCES OF A NEWLY FOUND pKT OF THIONINE ON THE MECHANISM IN BASIC SOLUTIONS

Abstract— Evidence for an acid-base equilibrium between the neutral and monocationic forms of the triplet state of thionine, ³ T and ³ TH ⁺ (p K _T# 8–9) has been found. This equilibrium, which has no counterpart in the methylene blue system can explain the different behaviours of these two similar dyes concerning their photoreduction by ethylene diamine tetracetic acid (EDTA) in basic solutions. With reasonable assumptions about the reactivity of the various species present in solution, it is now possible to calculate over the whole pH range the quantum yield of semireduced dye formation φs, which, according to the previously proposed mechanism, must be proportional to the overall photoreduction quantum yield φR. The very good fit between the calculated curve φs = f (pH) and the experimental one φR = f (pH) for both dyes strongly supports the proposed mechanism.     

Luminescence properties of acridine dyes in micellar sodium dodecyl sulfate solutions containing thallium ions

The effect of external heavy Tl^I ion on the luminescence properties of Trypaflavine, Acridine Yellow, and Acridine Orange solubilized in sodium dodecyl sulfate micelles was studied. An increase in the concentration of thallium ions results in a decrease in the intensity of prompt fluorescence, an increase in the intensity of delayed fluorescence, the appearance of phosphorescence at 20 °C, and a shortening of the triplet state lifetime of the dyes. The effective and micellar Stern—Volmer constants of fluorescence quenching of the dyes by thallium ions were determined. The effective and micellar quenching rate constants of triplet states of the dyes by Tl(i) ions and lifetimes of the triplet states of the dyes in the absence of thallium ions were calculated on the basis of kinetic measurements.     

DYE-SENSITIZED PHOTOLYSTS OF DISODIUM PHENYL PHOSPHATE IN AQUEOUS SOLUTION UNDER OXYGEN: INVOLVEMENT OF SINGLET OXYGEN

Abstract— The release of orthophosphate from oxygen-saturated aqueous solutions of disodium phenyl phosphate by near-UV and visible light was enhanced in the presence of the sensitizing dyes methylene blue, rose bengal and thionine. The reaction was accompanied by the bleaching of these dyes. In the absence of oxygen, under nitrogen, the photodecomposition was very much slower. In deuterium oxide as the solvent, the dye-sensitized photodecomposition was 9 times faster than in normal water. This result suggests that singlet oxygen is probably the reactive species in the dye-sensitized reaction.     

A THEORETICAL TREATMENT OF THE ELECTRONIC STATES OF THIONINE AND RELATED MOLECULES

Abstract— –Problems associated with the protolytic equilibria of thionine and related molecules in their lowest excited electronic states were investigated. The theoretical arguments are based on semi-empirical SCF MO (CI) calculations for the π-electronic system of these molecules; all singly excited configurations were included in the CI. The results indicate that the basic form of thionine in its ground, first excited singlet and lowest triplet state is protonated at the heterocyclic N atom. The difference of the p K values of these three states can be explained in terms of the calculated charge densities. The photochemical reactivity of the lowest triplet of the acidic form of thionine (³TH₂²⁺) differs greatly from that of the lowest triplet of the basic form (³TH⁺). Some arguments for the assignment of nπ* character to ³TH₂²⁺ and ππ* character to ³TH⁺ are advanced.     

STUDIES ON THYMINE AND URACIL TRIPLET EXCITED STATE IN ACETONITRILE AND WATER

Abstract— Thymine and uracil triplet-triplet absorption spectra and triplet excited state lifetimes have been observed in acetonitrile and water by nanosecond laser flash spectroscopy. A study of triplet energy transfer from these pyrimidines to retino! has also allowed an estimation of the triplet extinction coefficient ε_TT of thymine and uracil. These ε_TT were then used to determine the triplet quantum yields Ø_T of both pyrimidines in acetonitrile and water.     

ESTIMATION OF ENERGY DISTRIBUTION AND REDISTRIBUTION AMONG TWO PHOTOSYSTEMS USING PARALLEL MEASUREMENTS OF FLUORESCENCE LIFETIMES AND TRANSIENTS AT 77 K

Abstract— A single-sample method for estimating energy distribution and redistribution among the two photosystems using fluorescence lifetimes and transients at 77 K is presented. In this method,α(the fraction of photons absorbed by photosystem I, PSI) is F_1(α)/(F_1(α)+ (τ_{F 1(M)}/τ_{F 2(M)}).F_2(M)) where, F_1(α) is the fluorescence intensity from PSI excited by photons initially absorbed by the latter, τ_{F 1(M)} and τ_{F 2(M)} are the maximum lifetimes of fluorescence from chlorophyll- a in PSI (1) and II (2), and, F_2(M) is the maximum fluorescence intensity from PSII (P level). Analysis of the intensities and lifetimes of wavelength resolved fluorescence of thylakoids (pH 7.0), with and without cations, leads to the following conclusions: The addition of 10 m M Na⁺ to cation-depleted thylakoids (pH 7.0) increases α by ˜ 10%, while the subsequent addition of 10 m M Mg²⁺ leads to three principal concomitant changes (in the order of importance): a 50% decrease in PSII to PSI energy transfer, a 20% increase in other radiation-less losses, and a 10% decrease in α.     

Photophysical properties of the lowest excited singlet and triplet states of thio- and seleno-psoralens

The decay processes of the lowest excited singlet and triplet states of five heteropsoralens (HPS) were investigated by steady-state and shift-phase fluorometry and by laser-flash photolysis in different solvents. The emission spectra of HPS are detectable only in trifluoroethanol (TFE), where fluorescence lifetimes (τ_F) and quantum yields (φ_F) were measured. The triplet lifetimes (τ_T), triplet (φ_T) and singlet-oxygen production (φ_Δ) quantum yields were determined in benzene, ethanol and TFE by laser-flash photolysis. Semiempirical (INDO/1-CI) calculations allowed the nature of the lowest excited singlet and triplet states and transition probabilities to be obtained. Theoretical and experimental results indicate that the two lowest excited singlet states S₁ and S₂ of HPS are close-lying and different in nature (π,π* and n,π*). The "proximity effect" between these two states controls the photophysical properties of HPS as it does for the other furocoumarins. However, HPS have a peculiar behavior with respect to the related compounds because they are fluorescent and have, in three cases, detectable intersystem crossing only in TFE. This behavior can be tentatively explained by a different energy gap and/or order between the S₁ and S₂ states.     

Photochemistry of Macromolecular Dyes in Aqueous Solutions

Thiazine and phenazine dyes are known to exhibit photoredox behavior in the excited states of these molecules. These dyes are covalently attached to macromolecules, and the photochemistry has been investigated in homogeneous solution and as thin films coated onto electrodes. Flash photolysis of the macromolecular thionine in the presence of quenchers shows reduction of the dye and the subsequent disproportionation of the reduced dye. In the case of macromolecular thionine, evidence for the formation of a complex between ferrous ion and thionine is observed in the flash photolysis experiments. Photoelectrochemical studies show a new type of behavior for the macromolecular dye films coated onto electrodes. Macromolecular thionine film coated onto platinum electrode makes the electrode a cathode on illumination of the electrode. Macromolecular phenosafranine dye films coated onto electrodes change the polarity of the illuminated electrode depending upon the nature of the macromolecule.     

PHOTOPHYSICAL AND PHOTOSENSITIZING PROPERTIES OF 2-AMINO-4 PTERIDINONE: A NATURAL PIGMENT

Abstract— The characteristics of the fluorescence and phosphorescence emission of 2-amino-4 (3H) pteridinone (or pterin) in aqueous solutions are pH dependent. The room temperature fluorescence quantum yield is low and is maximum at pH = 10 (φ_F∼ 0.057). The 77K phosphorescence emission consists of two overlapping emissions originating from τ* triplet states. In agreement with low temperature results, the 353nm laser flash photolysis makes it possible to detect at pH 9.2, two transient triplet absorptions (τ₁∼ 0.3 μs and τ₂∼ 2.3 μs). The longer lived triplet is characterized by φ_TM∼ 0.20 and ∼ (550nm) = 2000 M ⁻¹ cm⁻¹. It reacts with the solvent forming the semireduced pterin with a quantum yield φ_R∼ 0.06. The photosensitizing properties of pterin have been studied by laser flash spectroscopy and steady state irradiations. Photoreactions implying singlet oxygen formation are shown to occur. Laser flash spectroscopy indicates that the pterin triplet is reduced by amino acids and nucleic acid bases. Corresponding bimolecular reaction rate constants have been measured.     

Spectral and photophysical studies of thiazine dyes in triton X-100

The absorption spectra of several thiazine dyes such as thionine, azure A, azure B, azure C and methylene blue in aqueous solution of Triton X-100 show that dyes as electron acceptors form 11 charge-transfer (CT) or electron-donor-acceptor (EDA) complexes with Triton X-100, which acts as an electron donor. From the thermodynamic and spectrophotometric properties of these complexes, the abilities of dyes to accept an electron are in the order: azure C > thionine > azure A > azure B > methylene blue. The photogalvanic effect in the aqueous solution dye-surfactant has been studied. Generation of photovoltage supports a CT or EDA interaction between thiazine dyes and Triton X-100. There is a good correlation among the photophysical (photovoltage), spectral and thermodynamic properties of these complexes.     

HETEROGENEITY IN THE THERMALLY-INDUCED QUENCHING OF THE PHOSPHORESCENCE OF MULTI-TRYPTOPHAN PROTEINS

Abstract— The steady-state intensity and lifetime of the tryptophan phosphorescence from a number of globular proteins in 2:1 (v/v) glycerol buffer were monitored as a function of temperature. The phosphorescence lifetimes are essentially independent of the tryptophan local environment in rigid solution at temperatures < 170K, but vary markedly between proteins at temperatures at which the solutions become fluid. The ratio of steady-state intensity to lifetime P/τ was found to be temperature independent despite the quenching for free tryptophan and the lone residue in myelin basic protein. Heterogeneity in the triplet quenching of the tryptophans in liver alcohol dehydrogenase and alkaline phosphatase were revealed as step-like decreases in the ratio of P/T followed by plateau regions characterizing the homogeneous behavior of the more resistent tryptophans in the proteins. This heterogeneity exists not only between solvent-exposed and buried residues, but reflects variations in the flexibility of the structure surrounding distinct buried tryptophans in the globular proteins.     

THE PHOTOSENSITIZED FORMATION AND REACTION OF SINGLET OXYGEN, O2*(1Δ), IN AQUEOUS MICELLAR SYSTEMS

Abstract— Oxygenated aqueous solutions of methylene blue containing dispersions of sodium dodecyl sulphate micelles with solubilised diphenyl isobenzofuran were irradiated with red light and the rate of loss of furan was followed over several min. The results are consistent with a mechanism in which singlet oxygen is produced by energy transfer from methylene blue triplet in the aqueous phase, and then diffuses to and penetrates the interior of the micelles where it reacts with the furan. The competition between this process and the natural decay of the excited oxygen has been examined and a β-value of 9.7 times 10^-5 M determined which is comparable to that for the same reaction in homogeneous solvents. A model of the system is proposed and its kinetic features discussed.     

Triplet state properties of croconate dyes in homogeneous and polymer-containing solutions

The effect of polymer microencagement on the lowest triplet excited state of croconate violet and croconate blue, members of a family of oxocarbon condensation derivatives which possess excellent electrocatalytic and photoelectrochemical dye sensitizing properties, has been investigated. These dyes, when surrounded by a poly(4-vinylpyridine) (PVP) matrix, displayed red-shifted S₀ → S₁ and T₁ → T_n absorption maxima. The triplet lifetimes for these dyes were 69 μs and 24 μs respectively in neat ethanol, and 134 μs and 36 μs respectively in ethanol containing 0.01 M PVP. The specific rates of triplet energy transfer from a triplet sensitizer to the dyes were lower in the polymer-containing solutions.