KINETIC PROPERTIES OF THE TRIPLET STATES OF METHYLENE BLUE AND OTHER PHOTOSENSITIZING DYES

Abstract— The long-lived (> 1 μsec) transients formed in the flash excitation of the representative photosensitizers methylene blue, eosin Y and pyrene have been investigated and various criteria have been used to distinguish between triplet state intermediates and chemical intermediates. Previous assignments of the triplet transients of methylene blue appeared less secure in view of the photochemical reactivity of this dye and its lack of phosphorescence. Earlier assignments of monomeric and dimeric triplet transients of methylene blue are substantiated, however, by the observations that the rate constant for quenching by oxygen is approximately 1/9^th diffusion controlled and the formation rates are commensurate with singlet decay rates and by the observation of triplet-triplet annihilation. Additional evidence in support of monomer triplet assignments for methylene blue and eosin Y is provided by the effect of heavy atom quenchers Cs⁺, Hg²⁺ and T1⁺ on decay rates. Due to chemical reactivity, quenching by I⁻appears less suitable as a diagnostic test for triplet state intermediates. The effect of N₃⁻, which is known to quench singlet oxygen molecules and to alter the course of photosensitized oxidations, on the triplet decay of methylene blue, eosin Y and pyrene is also investigated.     

THE CHARACTERISATION OF THE TRIPLET STATE OF CROCETIN, A WATER SOLUBLE CAROTENOID, BY NANOSECOND LASER FLASH PHOTOLYSES

Abstract— The triplet state of crocetin, which is a water soluble carotenoid, has been sensitized by psoralen. The triplet extinction coefficient, ε_T (73000 dm³ mol^-1 cm^-1 at 470 nm), the triplet-triplet spectrum and the quantum yield of triplet formation, φ_T (less than 1%) are reported in aqueous solution.
In order to calculate the extinction coefficient of crocetin it was necessary to obtain ε_T for psoralen in water (10000dm³ mol^-1 cm^-1 at 450 nm). This latter value was obtained using the complete conversion technique and is reported with the triplet-triplet spectrum.     

FLASH PHOTOLYSIS STUDIES OF OROTIC ACID

Abstract— The triplet state of orotic acid has been studied by flash photolysis. The rate for dimerization has been observed to vary from 2 × 10⁹ M ^-1 sec^-1 at pH 1 where both the triplet and ground state molecules are neutral, to under 10⁸ M^-1 sec^-1 above pH 9 where both the triplet and ground state molecules are doubly ionized. The p K of the triplet state has been measured as 4.6. The rate of oxygen quenching for the triplet is 2–3 × 10⁹ M^-1 sec^-1 while the rate of radiationless decay in solution is 0.73 × 10⁴ sec^-1. The triplet absorption spectra have been measured for the two ionic forms of the triplet.     

ENERGY CONVERSION IN THE DECAY OF TRIPLET LUMIFLAVIN IN THE PRESENCE OF FERRI- AND FERROCYANIDE

Abstract— Flash photolysis at 450 nm has been used to study the quenching of the excited triplet state of lumiflavin and the transient species formed in subsequent reactions in deaerated phosphate buffer (pH 6.9).
The effect of the presence of ferricyanide on the life time of triplet lumiflavin has been studied. The results suggest an energy transfer reaction without concurrent electron transfer reactions. The rate constant for the process was 2.8 times 10⁹ M ^-1 s^-1. The analogous reaction with ferrocyanide could not be observed because of the efficient electron transfer reaction (δG = -20.6 kcal mol^-1) leading to the formation of the semireduced lumiflavin and ferricyanide. The rate constant for this reaction was 3.3 times 10⁹ M ^-1 s^-1. The semireduced lumiflavin radical was found to disappear in a second order reaction with a rate constant of 1.7 times 10⁹ M ^-1 s^-1. It was found to react with ferricyanide with a rate constant of 0.7 times 10⁹ M ^-1 s^-1.
A model for the various photochemical and photophysical processes involved in the decay and quenching of the lumiflavin triplet state is suggested and discussed.     

TRIPLET LIFETIMES OF SOME FLAVINS

Abstract— Using flash photolysis the rate constants of the triplet decay at pH 7.0 (26°C) of 10-hydroxyethyl-isoalloxazine (2700 s⁻¹), 2'-deoxyRF (2300 s⁻¹), 5'-deoxyRF (3200 s⁻¹), 8-hydroxyRF (18 000 s⁻¹) and 8-aminoRF (28 000 s⁻¹) have been measured. The results agree with other evidence that photochemical properties of flavins are influenced by the interaction of the sidechain with the isoalloxazine nucleus. In addition, our data on RF and FMN indicate that the triplet decay rates given in the literature for these compounds have to be corrected to 3200 s⁻¹ (RF) and 4900 s⁻¹ (FMN), respectively. The rate constants for the quenching of the triplet by ground state molecules for all above compounds are given.     

QUENCHING OF CHEMIEXCITED TRIPLET ACETONE BY BIOLOGICALLY IMPORTANT COMPOUNDS IN AQUEOUS MEDIUM

Abstract— Thermolysis of tetramethyl-l,2-dioxetane is a convenient source of triplet acetone, which can be monitored in aerated solutions by the sensitized fluorescence of 9,10-dibromoanthracene. We have investigated the quenching of chemiexcited triplet acetone in air-equilibrated aqueous solutions containing the 9,10-dibromoanthracene-2-sulfonate ion by five classes of compounds: indoles, tyrosine derivatives, quinones, riboflavin, and xanthene dyes. Quenching rates for indoles, tyrosine and its 3,5-dihalogenoderivatives, and xanthene dyes (k_q= 10⁸-10⁹ M^-1 s^-1) are considerably smaller than the diffusion controlled rate, whereas those for quenchers with high electroaffinities, such as quinones (IP = 10–11 eV), approach the diffusion controlled rate (k_q= 10¹⁰ M^-1 s^-1). Energy transfer for riboflavin probably occurs by a triplet-singlet Förster type process.
A comparison of the present data with previous studies of quenching of enzymically generated triplet acetone (isobutanal/O₂/horseradish peroxidase) by the same classes of quenchers (except riboflavin) reveals that, independent of the nature of the quencher and the deactivation mechanism, the Stern-Volmer quenching constants ( k_q t⁰) are systematically about one order of magnitude higher in the enzymatic system. The difference is attributed to a longer lifetime of triplet acetone in the latter case, "protected" in an enzyme cavity against collisions with dissolved oxygen.     

THE TRIPLET AND RADICAL SPECIES OF HAEMATOPORPHYRIN AND SOME OF ITS DERIVATIVES

Abstract— Nanosecond laser flash photolysis and pulse radiolysis have been used to generate and characterise the triplet state, and semioxidised and semireduced radicals of haematoporphyrin, and three 0 -acyl compounds derived from it (the monoacetate, the diacetate and the disuccinate).
After 347 nm irradiation in water containing 2% Triton X-100, haematoporphyrin forms the triplet state (φ_T= 0.92) and photoionises monophotonically (φ_I= 0.03). For the O -acyl derivatives, φ_T approaches unity and photoionisation is reduced. In acetone the triplet yield of all four compounds are close to unity. The difference and corrected spectra for the triplet species are presented and decay rates ( k ₁˜10⁴s^-1) and oxygen quenching constants ( k _Q˜1.5times10⁹ M ^-1s^-1) for the triplet state have been measured. The difference and corrected spectra for the semi-reduced species in methanol and semi-oxidised species in aqueous Triton X-100 are presented.
The photophysical characteristics in fluid solution of haematoporphyrin and its 0 -acyl derivatives are rather similar to those previously recorded for other photosensitising porphyrins.     

TRANSIENT PHOTOCHEMISTRY OF NEUTRAL RED

Abstract— Flash photolysis of neutral red between pH 1.3 and pH 11 yields the triplet species ³DH₂⁺²³DH⁺ and ³D. Both ³DH₂⁺² and ³D exhibit first order decay with rate constants of 1.6 ± 0.3 × 10⁴ s^-1 but ³DH⁺ decays within the lifetime of the flash. Over the entire pH range, ascorbic acid quenches the triplet, forming the semireduced radicals DH₃⁺² DH₂⁺ and DH, all of which exhibit second order decay with k = 1.8 ± 0.4 ± 10⁸ M ^-1s^-1 most probably by recombination with semioxidized ascorbic acid. The dependence of the rate of decay of radical neutral red on the identity of reversible reductants supports the back-electron transfer mechanism, as does digital simulation of complex radical disproportionation schemes. In contrast to the efficient reduction of triplet neutral red by ascorbic acid, its reduction by EDTA is quite inefficient.     

QUENCHING OF CHLOROPHYLL FLUORESCENCE BY NITROBENZENE

Abstract—Nitrobenzene quenching of chlorophyll fluorescence in ethanol has been investigated. Steady state relative quantum yields have been measured and fluorescence decay rates were determined using both nanosecond photon counting and picosecond pulses from a mode-locked Nd³⁺ glass laser.
The fluorescence decay is described by
1( t )= I ₀ exp (- t/τ−At^1/2 )
the form predicted for decay governed by the kinetics of the continuum model of diffusion controlled reactions. From the parameters of the fluorescence decay, the encounter distance is 5–7 A° the mutual diffusion coefficient is 0.62 × 10^--⁵ cm2s^-1± 12%.
Some of the fluorescence quenching is also attributed to static quenching by a nitrobenzene-chlorophyll, ground-state complex. The equilibrium constant for formation of this ground-state complex was determined to be 4.1 M ^-1. The combined dynamic and static quenching model allows calculation of quantum yields of fluorescence in good agreement with the experimentally determined quantum yields.     

THE TRIPLET EXCITED STATES OF BILIVERDIN AND BILIVERDIN DIMETHYL ESTER

Abstract— The triplet states of biliverdin and biliverdin dimethyl ester have been generated using pulse radiolysis excitation. Biliverdin triplet was formed by energy transfer from biphenyl triplet in acetone, absorbed throughout the wavelength range studied (380–1000 nm) and had a half-life of 11.7μs under the cpnditions chosen. Biliverdin dimethyl ester triplet was formed by energy transfer from biphenyl triplet in benzene, likewise absorbed throughout the wavelength range studied (360–1000 nm) and had a half-life of 6.7μs under the conditions used. Both biliverdin and biliverdin dimethyl ester efficiently quench anthracene, naphthacene, but not μ-carotene, triplet states. On the other hand. neither μ-carotene nor oxygen were found to quench the triplet states of biliverdin or biliverdin dimethyl ester. Estimates or limits for the rate constants of all these quenching reactions were obtained. These reactivities suggest that the triplet levels of both biliverdin and biliverdin dimethyl ester lie around 90 kJ mol^-1. The triplet energy transfer rate from bilirubin to biliverdin dimethyl ester in benzene was measured to be 1.9 × 10⁹ M^-1 s^-1. The singlet-triplet intersystem crossing efficiencies of both molecules were very low, limits of 0.004 and 0.001 being found for biliverdin and biliverdin dimethyl ester, respectively, using 347 nm laser excitation.     

STUDIES ON SOME INTERMEDIATES AND PRODUCTS OF THE PHOTOREDUCTION OF 9,10-ANTHRAQUINONE *,†

Abstract— The mechanism of the photoreduction of 9,10-anthraquinone (AQ) in alcohol and hexane has been studied by flash photolysis. The fluorescence spectrum of the photoproduct, 9,10-dihydroxy anthracene shows a large shift between hexane and ethanol. The quantum yields of photoreduction for AQ are solvent-dependent, the reaction between the solvent radical and AQ determining the quantum yield.
The absorption spectrum of the 9,10-anthrasemiquinone (AQH.) has a long-wavelength absorption band with peaks at 631 and 678 nm. The second-order decay constants for AQH. were estimated to be 1.3 × 10⁹, 6.7 × 10⁸ and 2.0 × 10⁸ M ^-1 sec^-1 in ethanol, 2-propanol and ethylene glycol, respectively.
A long-wavelength absorption band was observed for 9,10-anthrasemiquinone radical anion, having peaks at 776 and 860 nm; epsi;_max= 1900 at 776 nm. This spectrum is compared with the spectra of 9,10-dihydroxy anthracene mono- and di-anions. The 9,10-anthrasemiquinone radical anion was found to photoreduce quantitatively to 9,10-dihydroxy anthracene mono-anion with a quantum yield of 0.1.     

THE PHOTOREDUCTION OF METHYLENE BLUE BY ARYLAMINOMETHANE-SULFONATES

Abstract— The photoreduction of methylene blue in the presence of arylaminomethanesulfonates (RAMS = RC₆H₄NHCH₂SO₃Na) was studied by laser and conventional flash photolysis. These compounds quenched the methylene blue triplet deviating from a normal Stern-Volmer behaviour. For low quencher concentrations, a Rehm-Weller relationship was found between the k _q's and the DL G 's obtained for the electron transfer reactions. The lack of further quenching at higher [RAMS] is ascribed to the formation of a ground state ion pair between the dye and the anionic quencher which, on excitation, forms a triplet state unable to under go electron transfer for steric reasons. A second order decay rate constant was found for the semireduced species (MB') ( ca. 5 × 10⁹ M _-1 s_-1, independent of the RAMS used) and is attributed to a proton transfer from the radical zwitterion (RC₆H₄NH CH₂SO₃⁻) to MB. The overall dependence on the substituent of the bleaching observed by continuous irradiation follows the triplet behaviour.     

ON THE MECHANISM OF QUENCHING OF SINGLET OXYGEN IN SOLUTION

Abstract— Bimolecular rate constants for the quenching of singlet oxygen O*₂(¹Δ_g), have been obtained for several transition-metal complexes and for β-carotene. Laser photolysis experiments of aerated solutions, in which triplet anthracene is produced and quenched by oxygen, yielding singlet oxygen which then sensitizes absorption due to triplet carotene, firmly establishes diffusion-controlled energy transfer from singlet oxygen as the quenching mechanism in the case of β-carotene. The efficient quenching of singlet oxygen by two trans-planar Schiff-base Ni(II) complexes, which have low-lying triplet ligand-field states, most probably also occurs as a result of electronic energy transfer, since an analogous Pd(II) complex and ferrocene, which both have lowest-lying triplet states at higher energies than the O*₂(¹Δ_g), state, quench much less effectively.     

THE IN VITRO PHOTOCHEMISTRY OF BIOLOGICAL MOLECULES-III. ABSORPTION SPECTRA, LIFETIMES AND RATES OF OXYGEN QUENCHING OF THE TRIPLET STATES OF β-CAROTENE, RETINAL AND RELATED POLYENES

Abstract— The triplet-triplet absorption spectra of six polyenes have been characterised using flash photolysis, in the presence of anthracene as sensitizer, and pulse radiolysis, in the absence of a sensitizer. The polyenes include several which contain carbonyl groups whose triplet states, unlike retinal , could not be detected unsensitized by flash photolysis. The triplet lifetimes appear to be a function of the number of conjugated double bonds, and vary between 7 and 14 μ sec. In general, the longer the polyene, the shorter the lifetime. An empirical linear relation was found between the frequencies of the polyene triplet-triplet absorption maxima, and the frequencies of the corresponding ground singlet-singlet maxima. The rate constants for quenching by oxygen of nine polyene triplet states were determined to lie in the range 2–7 × 10⁹ M ^-1 sec^-1. The possible mechanisms for oxygen quenching of triplet states are discussed and analogies between the results for oxygen quenching of polyenes and of polyacenes are drawn. The rate constant for oxygen quenching of all- trans -β-carotene triplet was the same in benzene and hexane.     

THE EFFECT OF CHEMICAL STRUCTURE ON THE PHOTOSENSITIZING EFFICIENCIES OF PORPHYRINS

Abstract— A kinetic investigation was performed on the photooxidation of methionine sensitized by various porphyrins at different oxygen concentrations. The rate of photooxidation was found to be strongly dependent on the nature of the sensitizer. In the case of hematoporphyrin, chelation of Mg²⁺ and Zn²⁺ and especially of Cu²⁺ and Fe²⁺ caused a significant decrease of the photosensitizing efficiency. Fluorescence and/or flash photolysis studies showed that such a decrease is ascribed to an enhancement of the non-radiative decay of the first excited singlet state as well as to a reduction of the triplet lifetime. The sensitizing efficiency is also dependent on the nature of the porphyrin side chains. A reaction mechanism involving ¹O₂ as the oxidizing agent is proposed.     

QUENCHING OF EXCITED TRIPLET LUMIFLAVIN BY LUMIFLAVIN RADICALS FORMED IN ITS T-T REACTION

Abstract— Reactions of the triplet state of lumiflavin (³LF) in water adjusted at pH 7.2 were reexamined by means of a Xe-flash photolysis and a laser photolysis. Measurements of the decay of ³LF were made on solutions of LF ranging in the concentration from 4 to 61 times 10^-6 mol/dm³. A one-electron reduced and a one-electron oxidized species of lumiflavin (LF^- and LF⁺) were produced in the first decay stage of ³LF with a high efficiency (0.6 ± 0.1) in a bimolecular triplet-triplet reaction. The product radicals (LFH^- and LF⁺) quench ³LF very efficiently (3 ± 0.8 × 10⁹ mol^-1dm³ s^-1) compared with LF in the ground state (> 2 × 10⁷ dm³ mol^-1).     

TRIPLET FORMATION AND TRIPLET DECAY IN REACTION CENTERS FROM THE PHOTOSYNTHETIC BACTERIUM Rhodopseudomonas sphaeroides

Abstract— In the reaction center of photosynthetic bacteria, with the primary ubiquinone reduced, the triplet state P^R of the primary electron donor (a pair of bacteriochlorophylls named P) is PO ulated with a takes place in a few ns. We measured by flash absorption spectroscopy the influence of temperature on formation and decay kinetics of P^R and ³Car in the reaction center of several strains of R. sphaeroides . The rate of triplet energy transfer, measured as the decay of P^R after a flash, decreases when the temperature is lowered. Between 60 and 30 K the half-time of energy transfer becomes longer than the ³Car half-time decay (about 6 μs) and below 20 K the transfer is slower than the internal decay of P^R (about 100 μs). In several cases it is clear that P^R and ³Car decay independently and are not in thermal equilibrium. The singlet energy transfer from carotenoid to P occurs with a high efficiency at all temperatures.
The data can be accounted for on the basis of estimated energy levels of P^R and ³Car, in the context of the equilibrium ³P ←³D where ³P is the localized triplet state of P-870 and ³D is another triplet state. A reasonable kinetic scheme leads us to estimate that ³D is 0.0025 ± 0.005 eV above ³P. ³D may thus be the state observed by Shuvalov and Parson (1981). We propose that both triplet and singlet energy transfer between P and the carotenoid occur via a bacteriochlorophyll, to which the carotenoid should be tightly coupled via exchange interaction.     

ACTINOMETRY IN MONOCHROMATIC FLASH PHOTOLYSIS: THE EXTINCTION COEFFICIENT OF TRIPLET BENZOPHENONE AND QUANTUM YIELD OF TRIPLET ZINC TETRAPHENYL PORPHYRIN

Abstract— The extinction coefficient ε_T, of triplet benzophenone in benzene has been directly determined by absolute measurements of absorbed energy and triplet absorbance, Δ D ⁰_T, under demonstrably linear conditions where incident excitation energy, E ₀, and ground state absorbance, A ₀, are both extrapolated to zero. The result, 7220 ± 320 M ^-1 cm^-1 at 530 nm, validates and slightly corrects many measurements relative to benzophenone of triplet extinction coefficients made by the energy transfer technique, and of triplet yields obtained by the comparative method.
As E ₀ and A ₀ both decrease, Δ D ⁰_T becomes proportional to their product. In this situation, the ratio R = (1/ A ₀)(dΔ D ⁰_T/d E ₀) = (ε_T - ε_G)φ_T. Measurements of R , referred to benzophenone, give (ε_T - ε_G)φ_T for any substance, without necessity for absolute energy calibration.
Both absolute and relative laser flash measurements on zinc tetraphenyl porphyrin (ε_T - ε_G at 470 nm = 7.3 × 10⁴ M ^-1 cm^-1) give φ_T= 0.83 ± 0.04.     

ELECTRON TRANSFER FROM THE EXCITED STATE OF TYROSINE TO COMPOUNDS CONTAINING DISULFIDE LINKAGES

Abstract— The flash photolysis of aqueous solutions of tyrosine has been studied in the presence of various concentrations of the cyclic disulfide sodium lipoate (thioctic acid, Na⁺ salt). In addition to the formation of phenoxyl radicals and hydrated electrons (and possibly H atoms) from the photoionization of tyrosine, the characteristic spectrum of the radical anion RSSR^- of lipoate was also observed in neutral as well as in alkaline solutions. From the dependence of these yields upon the concentration of lipoate, it was found that a long–lived triplet excited state of tyrosine, rather than the singlet excited state, is involved in these reactions. The negative radical ions RSSR^- are formed by two distinct pathways: (a) Na⁺–lipoate reacts with the solvated electrons which are ejected from the tyrosine triplets ³Tyr → RO.+ e ^-_aq+ H⁺ followed by e ^-_aq+ RSSR → RSSR^-, and (b) by direct interaction of lipoate with triplet excited tyrosine, resulting in the transfer of a negative charge from tyrosine to the disulfide linkage. At high lipoate concentrations, the singlet excited state of lipoate is quenched, k ₄= 1.6 × 10¹⁰ M ^-1 sec^-1, but this reaction does not lead to the formation of RSSR^- radical ions.     

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).