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Laser photochemistry of oxygen. Application to studies of the absorption spectra of dissolved oxygen molecules
Alexander Krasnovsky1,2*, and Anton Kozlov1
1 Federal Centre for Biotechnology, A.N. Bach Institute of Biochemistry, Russian Academy of Science, 33 Leninskii pr., Moscow 119071, Russian Federation
2 M.V. Lomonosov Moscow State University, Department of Biology, Vorobyevy gori, Moscow 119899, Russian Federation
* e-mail: phoal@mail.ru
Abstract. This paper summarizes the data of our lab on the rates of photooxygenation of singlet oxygen traps upon direct laser excitation of oxygen in air-saturated organic solvents and water. Methods of application of these data to calculation of absorbance (4) and molar absorption coefficients (e) in the maxima of the main oxygen absorption bands (1273, 765 and 1070 nm) are discussed. The most accurate results were obtained from comparing the photooxygenation rates upon porphyrin-photosensitized and direct excitation of oxygen molecules. It is shown that £1273 is not sensitive to the presence of heavy atom (bromine) in solvent molecules and markedly decreases on going from nonpolar solvents to water being proportional to the radiative rate constants obtained from the quantum yields of singlet oxygen phosphorescence at 1274 nm. The coefficient £765 markedly increases in the presence of bromine. In solvents lacking heavy atoms the 1.52-fold increase of £765 was observed on going from non-polar solvents to water. Simultaneously, the ratios £1273/£765 are changed from (7-10)/1 in non-polar solvents to 1.5/1 in water. The value of £1070 obtained in carbon tetrachloride is shown to be about two orders smaller than £1273 in the same solvent. The results are important for both analyses of oxygen photonics and dosimetry of laser radiation in biomedical experiments. © 2017 Journal of Biomedical Photonics & Engineering.
Keywords: Molecular oxygen; singlet oxygen; photosensitizers; direct oxygen excitation.
Paper #3171 received 12 Mar 2017; revised manuscript received 1 Apr 2017; accepted for publication 1 Apr 2017; published online 4 Apr 2017. doi: 10.18287/JBPE17.03.010302. [Special Issue. Years in Biophotonics: 70th Anniversary of Prof. A.V. Priezzhev].
References
1. R. S. Mulliken, "Interpretation of the Atmospheric Oxygen Bands: Electronic levels of oxygen molecules," Nature. 122(3075), 505 (1928).
2. R. S. Mulliken, "Interpretation of the atmospheric absorption bands of oxygen," Phys. Rev. 32(6), 880-887 (1928).
3. G. Herzberg, "Photography of infrared solar spectrum to wavelength 12,900 A," Nature 133(3368), 759 (1934).
4. L. Herzberg, and G. Herzberg, "Fine structure of the infrared atmospheric oxygen bands," Astrophys. J. 105, 353-359 (1947).
5. V. D. Galkin, "Electronic transition moment of the b:E+g - X3E-g system of oxygen bands," Opt. Spektr. 47(2), 266-270 (1979) [in Russian].
6. V. I. Dianov-Klokov, "On the absorption spectrum of condensed oxygen in the region of 1.27-0.3 micrometers," Opt. Spektr. 20, 954-962 (1966) [in Russian].
7. D. F. Evans, "Oxidation by photochemically produced singlet states of oxygen," Chem. Comm., 367-368 (1969).
8. I. B. C. Matheson, and J. Lee, "Comparison of the pressure dependence of the visible and infrared electronic absorption spectra of oxygen in gas and in Freon solution," Chem. Phys. Lett. 8(2), 173-176 (1971).
9. C. Long, and D.R. Kearns, "Selection rules for the intermolecular enhancement of spin forbidden transitions in molecular oxygen," J. Chem. Phys. 59, 5729-5736 (1973).
10. A. U. Khan, "Singlet molecular oxygen spectroscopy: chemical and photosensitized," in Singlet O2, vol. 1, A. A. Frimer (ed.), CRC Press, Boca Ratoh, Florida, 39-176 (1985).
11. G. P. Gurinovich, "Molecular-oxygen photonics," J. Applied Spectrosc. 54(3), 403-411 (1991).
12. I. B. C. Matheson, and J. Lee, "Reaction of chemical acceptors with singlet oxygen produced by direct laser excitation," Chem. Phys. Lett. 77, 475-476 (1970).
13. I. B. C. Matheson, and J. Lee, "Chemical reactions rates of aminoacids with singlet oxygen," Photochem. Photobiol. 29(5), 879-881 (1979).
14. R. V. Ambartzumian, "Lasers in cardiology," Proc. SPIE. 701, 341-343 (1987).
15. R. V. Ambartzumian, P. G. Eliseev, B. V. Eremeev et al., "Biological effects of laser radiation on erythrocytes in the infrared absorption band of molecular oxygen," Short Comm. In Physics, P. N. Lebedev Physics Institute RAS, 35-37 (1987) [in Russian].
16. S. D. Zakharov, and A. V. Ivanov, "Light-oxygen effect in cells and its potential applications in tumor therapy," Quantum electronics. 29(12), 1031-1053 (1999).
17. T. Karu, "Primary and secondary mechanisms of action of visible to near-IR radiation on cells," J. Photochem. Photobiol. B 49, 1-17 (1999).
18. Yu. A. Vladimirov, A. N. Osipov, and G. I. Klebanov, "Photobiological principles of therapeutic applications of laser radiation," Biochemistry (Moscow) 69(1), 81-90 (2004).
19. A. S. Yusupov, S. E. Goncharov, I. D. Zalevskii, V. M. Paramonov, A.S. Kurkov, "Raman fiber laser for the drug-free photodynamic therapy," Laser Physics 20(2), 357-359 (2010).
20. A. A. Krasnovsky, Jr, N. N. Drozdova, A. V. Ivanov, and R. V. Ambartzumian, "Activation of molecular oxygen by infrared laser radiation in pigment-free aerobic systems," Biochemistry (Moscow) 68(9), 963-966 (2003).
21. A. A Krasnovsky, Jr., R. V. Ambartzumian, "Tetracene oxygenation caused by infrared excitation of molecular oxygen in air-saturated solutions. The photoreaction action spectrum and spectroscopic parameters of the 1Ag ^ 3Sg - transition in oxygen molecules," Chem. Phys. Lett. 400(4-6), 531-535 (2004).
22. A. A. Krasnovsky, Jr., N. N. Drozdova, Ya. V. Roumbal, A. V. Ivanov, and R. V Ambartzumian, "Biophotonics of molecular oxygen: activation efficiencies upon direct and photosensitized excitation," Chinese Opt. Letters 3(S1), S1-S4 (2005).
23. A. A. Krasnovsky Jr., Ya. V. Roumbal, A. V. Ivanov, and R. V. Ambartzumian, "Solvent dependence of the steady-state rate of :O2 generation upon excitation of dissolved oxygen by cw 1267 nm laser radiation in air-saturated solutions. Estimates of the absorbance and molar absorption coefficients of oxygen at the excitation wavelength," Chem. Phys. Lett. 430(4-6), 260-264 (2006).
24. A. A. Krasnovsky, Jr., I. V. Kryukov, and A. V. Sharkov, "Photooxygenation of singlet oxygen traps upon excitation of molecular oxygen by dark red laser radiation in air-saturated solutions," Proc. SPIE 6535, 65351Q (2007).
25. A. A Krasnovsky, Jr., Ya. V. Roumbal, and A. A. Strizhakov, "Rates of :O2 (:Ag) production upon direct excitation of molecular oxygen by 1270 nm laser radiation in air-saturated alcohols and micellar aqueous dispersions," Chem. Phys. Lett. 458(1-3), 195-199 (2008).
26. A. A. Krasnovsky, Jr., A. S. Kozlov, and Ya.V. Roumbal, "Photochemical investigation of the IR absorption bands of molecular oxygen in organic and aqueous environment," Photochem. Photobiol. Sci. 11(6), 988-997 (2012).
27. A. A. Krasnovsky, Jr., and A. S. Kozlov, "New approach to measurement of IR absorption spectra of dissolved oxygen molecules based on photochemical activity of oxygen upon direct laser excitation," Biophysics 59(2), 199-205 (2014).
28. A. A. Krasnovsky, Jr., and A.S. Kozlov. "Photonics of dissolved oxygen molecules. Comparison of the rates of direct and photosensitized excitation of oxygen and reevaluation of the oxygen absorption coefficients," J. Photochemistry and Photobiology, A: Chemistry 329, 167-174 (2016).
29. F. Anquez, E. Courtade, A. Sivery, and S. Randoux. "A high-power fiber ring laser for the investigation of singlet oxygen production from direct laser excitation around 1270 nm," Optics Express 18(22), 22928-22936 (2010).
30. A. Sivery, A. Barras, R. Boukherroub, C. Pierlot, J. M. Aubry, F. Anquez, and E. Courtade, "Production rate and reactivity of singlet oxygen 1O2(1Ag) directly photoactivated at 1270 nm in lipid nanocapsules dispersed in water," J. Phys. Chem. C 118(5), 2885-2893 (2014).
31. M. Bregnh0j, M. V. Kr£gp0th, R. J. Serensen, M. Westberg, P. R. Ogilby, "Solvent and heavy-atom effects on the O2(X3£g-) ^ O2(b1Eg+) absorption transition," J. Phys. Chem. A 120(42), 8285-8296 (2016).
32. S. L. Murov, I. Charmichael, and G. L. Hug, Handbook of Photochemistry, Marcel Dekker Inc., New York, Basel, Hong Kong, 290 (1993).
33. M. Hild, and R. Schmidt, "The mechanism of the collision-induced enhancement of the a ^ X3Zg- and radiative transitions of O2," J. Phys. Chem. A 103(31), 6091-6096 (1999).
34. B. F. Minaev, "Electronic mechanisms of activation of molecular oxygen," Russian Chem. Rev. 76(11), 10591083 (2007).
35. B. F. Minaev, "Spin-orbit coupling mechanism of singlet oxygen a1Ag quenching by solvent vibrations," Chemical Physics 483-484, 84-95 (2017).
36. S. J. Strickler, and R. A. Berg, "Relationship between absorption and intensity and fluorescence lifetime of molecules," J. Chem. Phys. 37(4), 814-822 (1962).
37. A. A. Krasnovsky, Jr., "Photoluminescence of singlet oxygen in pigment solutions," Photo-chem. Photobiol. 29(1), 29-36 (1979).
38. A. A. Krasnovsky, Jr., "Singlet molecular oxygen and primary mechanisms of photodynamic action of optical radiation.," In Reviews on Science and Technology, Modern Problems of Laser Physics., S. A. Akhmanov, and E. B. Chernyaeva (eds.), All Union Institute of Science and Technology Information (VINITI), Moscow, vol. 3, 63-135 (1990) [in Russian].
39. A. A. Krasnovsky, Jr., "Primary mechanisms of photoactivation of molecular oxygen. History of development and the modern status of research," Biochemistry Moscow 72(10), 1065-1080 (2007).
40. F. Anquez, I. E. Yazidi-Belkoura, S. Randoux, P. Suret, and E. Courtade, "Cancerous cell death from sensitizer free photoactivation of singlet oxygen," Photochem. Photobiol. 88(1), 167-174 (2012).
41. L. O. Klotz, "Oxidant-induced signaling: Effects of Peroxynitrite and singlet oxygen," Biol. Chem. 383(3-4), 443-456 (2002).
42. L. O. Klotz, K-D. Kronke, and H. Sies, "Singlet oxygen-induced signaling effects in mammalian cells," Photochem. Photobiol. Sci. 2(2), 88-94 (2003).
43. R. J. Antony, K. L. Warczak, and T. J. Donohue, "A transcriptional response to singlet oxygen, a toxic byproduct of photosynthesis," Proc. Natl. Acad. Sci. USA 102(18), 6502-6507 (2005).
44. R. V. Ambartzumian, "Selective application of laser light with wavelength 1268 ^m for treating solid tumours. Physical methods for potentiating the immune response," Laser medicine 20(2), 62-63 (2016) [in Russian].
1 Introduction
It has been known since the seminal papers of R. Mulliken that oxygen molecules have triplet ground state and two relatively low-lying singlet levels [1,2]. Fig. 1 shows energy diagram of the major electronic transitions in monomeric oxygen molecules in rarified gas as follows from measurements of the absorption spectra in Earth atmosphere and in the gas phase at atmospheric pressure [3-6]. These transitions are highly forbidden therefore the absorption bands of monomeric oxygen molecules are so weak that they normally cannot be measured using conventional spectrophotometers under ambient conditions.
Reliable absorption spectra were obtained in the gas phase and solutions under high oxygen pressure of 50140 atm and in liquid oxygen. However, under these conditions dimerization of oxygen molecule occurs, therefore the absorption spectra correspond mainly to dimols (O2)2. In the dimol spectra, the IR maxima analogous to those of monomols are shifted toward shorter wavelengths, and a series of additional maxima appear in visible region. Relative intensities of the spectral peaks of dimols are strongly different from those in the gas phase at atmospheric pressure [6-11] (Fig. 2).
lo2 i'VO)]
4>г [4,(1)1
10; [4(0)]
S s ж s - п С к С 1-ц.
ж г-- 1-1
30: [%~(0)]
Fig. 1 The main electronic transitions in monomeric oxygen molecules in the rarefied gas as reported in refs [3-6]. The vibrational sublevels are shown in brackets.
It was reported in the classic papers by Evans [7] and Matheson's group [12,13] that excitation of oxygen dimols dissolved in organic solvents at oxygen pressure 130-140 atm. leads to formation of the :Ag singlet oxygen, which causes oxygenation of added organic
singlet oxygen traps, aminoacids, proteins and other molecules.
Fig. 2 Absorption spectrum of oxygen dimols in compressed oxygen at 150 atm. in the 5 cm optical path [10,11].
Based on these experiments, Ambartzumian and his collaborators advanced an idea that direct laser excitation of intrinsic dimol and monomol oxygen molecules in living cells might be a reason for biological action of laser radiation in visible and infrared [14,15]. It was also suggested that laser excitation of oxygen might be applied to destruction of cancer cells in vitro and in animal and human organisms.
These papers caused heated discussions. Apparently, biological action caused by laser excitation of oxygen molecules should be directly proportional to the rate of :O2 production by laser radiation, which in turn, depends on laser power (haS) and the fraction of laser light absorbed by oxygen molecules (1-10-Aox), where Aoxis oxygen absorbance (Aox << 0.01). Hence:
Biological effect ~ ~ has (1-10Aox) = has X 2.3 X Ao
(1)
Although biological and therapeutic action of red and IR lasers has been reported many times ([15-19] and refs therein) there is no crucial evidence showing that this action under ambient conditions and moderate (therapeutic) laser power is always due to oxygen excitation. Thus, reliable information on absorption coefficients of oxygen molecules under normal conditions is of great importance for both understanding of oxygen photonics in natural environment and mechanistic analyses of laser action and dosimetry of IR laser radiation in biomedical experiments.
Several years ago our group started work on mimicking biological effects of laser radiation in solutions of singlet (1Ag) oxygen traps in organic solvents and water at normal pressure and temperature. It was established that monomeric oxygen molecules dissolved in these solvents work as photosensitizers of oxygenation reactions. Kinetic analyses of the reaction rates allowed us to develop methods for calculation of absorbance (optical density) for oxygen absorption bands under natural conditions. The present paper summarizes major results of our group [20-28].
2 Experimental procedure
For sample irradiation, diode lasers LAMI Gelios ("Surgical innovation technologies", Moscow) were applied with the maxima of the emission bands at 12671270, 1064-1070 and 760-762 nm and the half bandwidth 4-12 nm. Laser light was focused into a fiber light guide with of 1 mm diameter. The eliciting light power, was controlled by the power meter Ophir ORION-TH with the sensor head 20C-SH (Israel). Solutions were irradiated in the 10 mm rectangular quartz cell under normal pressure and room temperature. Volume of the solutions was 1.5 ml. Diameter of the irradiated spot was 5-8 mm.
Tetracene, (Aldrich Chemistry, >98%), 1,3-diphenylisobenzofuran (DPIBF) and uric acid (Acros Organics) were used as singlet oxygen traps. Tetraphenylporphyrin (TPP) (Aldrich, Chemistry) was employed in certain experiments as photosensitizers of singlet oxygen production. Absorption spectra were measured using a Hitachi U-3400 (Japan) and SF-56 (LOMO Spektr, St. Petersburg) spectrophotometers. The rate of :O2 formation was calculated from the rate of photobleaching of the main absorption maxima of the traps (474 nm for tetracene, 410-414 nm for DPIBF, 530 nm for rubrene and 290 nm for uric acid) under irradiation by IR lasers.
It was shown that irradiation of air-saturated solutions of the traps in organic solvents and water by IR lasers of moderate power (0.03-2 W) with the emission maxima at 1270, 1070 and 765 nm causes oxygenation of the traps, which is accompanied by bleaching of their main absorption bands. The oxygenation rates were measured as follows. At first, the absorbance (optical density) (A0) in the main absorption maximum of a trap was recorded. In most experiments, solutions with A0 = 0.8-1.5 in the 1 cm cell quartz were employed. Then, the solutions were irradiated during 1-30 min, shaken, and the absorbance (Af) was measured again. The photoreaction rates (Viasr, AA/min) were calculated from measuring the illumination time (t) needed to decrease the absorbance so that AA = A0 - Af = 0.05 - 0.1. As a result, not more than 15% overall trap content was oxidized during irradiation. The oxygenation rates were then normalized to power of laser radiation in einstein/s (see details in refs [20-28]).
2.1 Mechanistic analyses
The photooxygenation action spectra were measured using tunable lasers. These spectra indicate that oxygenation of the traps occurred due to direct laser excitation of oxygen molecules. In CCl4, C6F6 and ethanol the main IR absorption maximum was found at 1273 nm. The width (FWHM) of this band was 17-18 nm (106 cm-1) [20,21,23] (Fig. 3). This maximum is shifted by 5 nm to longer wavelengths compared to the absorption maxima of monomeric oxygen in rarified gas (Fig. 1). More recently these results were confirmed by
Wavelength, nm
Fig. 3 Action spectra of oxygenation of the singlet oxygen traps under direct excitation of oxygen molecules by IR laser radiation. A: spectra obtained using tunable titan-sapphire laser in solutions of tetracene (•) [24] and 1,3-diphenylisobenzofuran (□) in CCU [26]. B: spectra obtained using tunable forsterite laser in solutions of tetracene in CCl4 (•) [21] and 1,3-diphenylisobenzofuran in ethanol (□) [23]. Vr is the rate of trap photooxygenation, n is irradiation intensity in number of photons per second.
Courtade's group in France who observed the oxygen excitation band at 1273 nm in CCU, acetone, ethanol and D2O with FWHM ~15 nm [29,30].
The action spectra in the dark red region were reported for the first time in papers of our group [24,26] (Fig. 3). In CCl4, C6F6 and ethanol the maximum was found at 765 nm, at slightly longer wavelengths than in the rarefied gas (Fig. 1). The band width was estimated to be 8-9 nm (~130 cm-1). Recently Ogilby's group in Denmark confirmed main results of our studies using luminescence technique and extended measurements to several additional solvents. In the luminescence experiments of this group the excitation maxima varied within 763-772 nm and FWHM -within 6-17 nm. In agreement with our data in CCl4 they observed the excitation maximum at 765 nm with the bandwidth of 7 nm (120 cm-1) [31]. Apparently, parameters of the action spectra characterize the major absorption bands of dissolved oxygen under ambient conditions.
In organic solvents the oxygenation of the traps was inhibited by singlet oxygen quencher - p-carotene, in water and alcohols - by sodium azide. The photoreaction was not observed after purging with argon or nitrogen. After saturation with pure oxygen the photoreaction rates increased 5-fold compared to air-saturated solutions [20-24]. Thus, evidence was obtained that under ambient conditions, monomeric oxygen molecules work as photosensitizers of oxygenation reactions similarly to oxygen dimols appearing at high oxygen pressure. The mechanisms of the photoreactions correspond to the following scheme:
O2 [3Eg"(F=0)] + hv765 ^ !O2 [lZg+(^=0)] ^
^ 1O2 [1Ag(^=0)]>
1Û2(1Ag) + Trap ^ oxygenation.
According to this scheme, photoexcitation of oxygen at 1273 nm causes direct population of the [1Ag(v=0)] (v is a vibrational sublevel) state. Photoexcitation at 1070 nm causes direct population of the [1Ag(v=1)] state and photoexcitation at 765 nm causes population of the singlet level 1Zg+(v=0). It is known that in the solution-phase [1Ag(v=1)] and 1Zg+(v=0) rapidly (for nano- or picoseconds) lose extra energy and populate the 1Ag(v=0) state due to collisions of oxygen and solvent molecules ([19-21]). Thus, no matter what level of oxygen is initially populated, chemical activity is determined by the 1 Ag(v=0) state of O2.
2000 ■
H
= 1500 ■
! ■
£ 1000 ■
1 ■
- 500 -
0 -—-.--.--.
1230 1270 1310
Wavelength, nm
Fig. 4 Spectrum of 1O2 phosphorescence in air-saturated pigment-free C6F6 upon excitation of dissolved oxygen by monochromatic light of cw diode laser at 766 nm. The spectrum was measured using three changeable interference filters transmitting light at 1230, 1270 and 1310 nm respectively. The luminescence data were obtained in collaboration with A.S. Benditkis.
O2 [3Eg-(^=0)j + ÎIV1070 ^ 1O2 [1Ag(^=1)j ^ ^ 1O2 [1Ag(^=0)]>
O2 [3Eg-(^=0)] + hV1273 ^ 1O2 [1Ag(^=0)]>
Quite recently we assembled a very sensitive spectrometer, in which phosphorescence of ^2 at 1270 nm is detected using cooled S-1 PMT (FEU-112) through three changeable interference filters with the transmission maxima at 1230, 1270 and 1310 nm. In
agreement with the above scheme and the data of Ogilby's group [31] we observed singlet oxygen phosphorescence at 1270 nm in air-saturated CCl4 and CôFô under excitation by cw laser at 765 nm (900 mW) without pigments-photosensitizers. First results are shown in Fig. 4. No light emission was observed upon excitation at 740 nm. Addition of acetone (50%) caused the decrease of luminescence intensity by more than two orders. Under oxygen purging the luminescence intensity increased (more detailed paper is in preparation).
2.2 Principles of data analysis
Based on this mechanistic scheme, the following equation was proposed for the rate (Viasr, M/s) of the trap photooxygenation under laser excitation of oxygen [27-28]:
Viasr = 23AmaxIiasakox[Trap]/ (k& + kox[Trap\), (2)
where Amax is absorbance (optical density) in the maxima of the oxygen absorption spectrum, Ia is the incident photon flux of IR laser radiation in einsteinxL~ls~1, koxis the rate constant of oxygenation of the traps by singlet oxygen; [Trap] is the molar concentration of the trap; kA is the rate constant of the 102 deactivation in the trap-free solutions, i.e. kA = 1/ta where ta is the lifetime of singlet oxygen in solvents lacking the singlet oxygen traps.
The coefficient a corresponds to degree of overlapping of the action spectrum with the spectrum of laser radiation. It was estimated from the photooxygenation action spectra (Fig. 3) and laser emission spectra using the "Origin" program:
a = ¡A(v)I(v)dv)/i I(v)dv,
(3)
where A(v) is a function characterizing normalized excitation spectrum of oxygen (Fig. 2) in energy units, in which A varies from 0 to 1; and I(v) is a function characterizing the normalized spectrum of laser radiation, where I varies from 0 to 1. Apparently, if the wavelength of laser light exactly corresponds to the maximum of the oxygen absorption band, a is equal to 1. Usually, the emission maxima of diode lasers, were shifted to shorter wavelengths compared to the oxygen absorption maxima, therefore a varied from 0.2 to 0.9 (see [27,28] for details).
Eq. 2 allows for accurate determination of the oxygen absorption coefficients corresponding to the maxima of oxygen absorption bands using experimental values for the oxygenation rates under laser irradiation:
Amax = Viasr(k& + koX[ Trap])/2.3alias koX[ Trap]). (4)
Knowing Amax, one can calculate molar absorption coefficients of oxygen Smax = AmaJ[Oi\, where [O2] is the molar concentration of oxygen in solutions. The
absorption cross section Umax = 2300Smax/ Nav where Nav is Avogadro number.
2.3 Absorbance at 1273 nm
The first paper, in which A 1273 was estimated using Eq. 4 and air-saturated carbon tetrachloride as a solvent, was published in 2004 [25]. The data were obtained from measurement of the oxygenation rates of tetracene upon excitation of dissolved oxygen at 1273 nm by tunable forsterite laser. The authors arrived to the values: A1273 « 7.2x10-6, £1273 « 0.003 M-1cm-1, 01273 «10-23 cm2, which served us as a reference during several years. Because of technical limitations higher precision could not be reached at that time. Then, accurate measurements of the relative rates of DPIBF oxygenation in different solvents were carried out. The results allowed analysis of the solvent dependence of the oxygen absorption coefficients. The first paper on this subject was published in 2005 [22]. Later, more detailed papers followed [23,25]. In particular, it was established that in accord with the Einstein law, the molar absorption coefficients corresponding to the oxygen absorption band at 1273 nm in series of organic solvents were proportional to the radiative rate constants (kr) for this electronic transition obtained from measurement of the quantum yield of singlet oxygen phosphorescence (n is the refractive index) [22-25]:
£1273 ~ kr/ n2.
(5)
However, in the papers mentioned above the proportionality was not revealed in water and alcohols. We proposed that this effect might be due to problems in kinetic analyses of the data [26,27]. Indeed, in the first papers we assumed that the rate constant (kox) for the reaction of the trap with 1O2 and molar absorption coefficients of traps (etmp), which were used for determination of the trap concentrations, did not depend on solvents. In order to solve this problem an experimental procedure was developed, which excluded etrap and kox from calculations. To reach this goal, the rates of oxygenation of the traps were compared upon direct and photosensitized oxygen excitation at equal trap concentrations. In this case, the following equation was obtained [26-28]:
Amax = ( Viasr/ Vpsr)(Ipsex/alias) 0a(1-1O-aPs)/2.3, (6)
where Vpsr is the rate of photosensitized oxygenation of the traps; Ia and Ipsex are the incident photon flux in einstein L~l s"1 for light applied to excitation of oxygen and photosensitizer respectively; Oa is the yield of singlet oxygen generation by pigments photosensitizers (TPP and TPPS were employed in our experiments). For calculations with Eq. 6, the photooxygenation rates were measured in the AA/t units, where AA is a change of absorbance in the maxima of the absorption spectra of the traps and t is time (minutes) of laser irradiation. Instead, one has to know accurate values of Oa for
Table 1 Values of the oxygen absorption coefficients in several solvents calculated according to Eqs. 4 and 6, using the rates of oxygenation of the singlet oxygen traps under ambient conditions.
A1273 *105 (1 cm) ^765 x105 61273X103, 6765 x103, 6765/ Permit_(1 cm)_ mM M-1cm-1 M-1cm-1 61273 tivity
Eq. 4* Eq. 6** Eq. 6** [32]
CCl4 - 1.33 0.19 2.6 5.1 0.7 0.14 2.24
CôFÔ 2.25 2.25 0.34 4.4 5.1 0.77 0.15 2.05
CôHô 1.2 1.20 0.13 1.9 6.3 0.68 0.11 2.27
Toluene 1.2 1.18 0.12 1.8 6.5 0.67 0.10 2.38
n-heptane - 0.92 0.15 2.8 3.3 0.53 0.16 1.94
1-Bromo-hexane - 0.96 0.36 3.0 3.2 1.2 0.38 5.4
Acetone 0.70 0.66 0.20 2.4 2.8 0.83 0.30 20,7
Ethanol - 0.48 0.14 1.65 2.9 0.85 0.29 24.5
H2O - 0.043 0.032 0.29 1.5 1.1 0.75 80.1
*The data of this column were obtained using Eq. 4 from comparison of the rates of DPBIF oxygenation in organic solvents and carbon tetrachloride. The absorption coefficients in CCI4 were measured using Eq. 6 (see [28] and refs therein). **The numbers in these columns (new data of our experiments) were obtained using Eq. 6 from comparison of the oxygenation rates upon direct and photosensitized oxygen excitation in each solvent. Maximum deviation from the average values indicated in this table did not exceed 10%.
photosensitizers. Our experiments show that for monomeric TPP and TPPS in most solvents used in our work, Oa = 0.73±0.05 ([26-28] and refs therein), only in 1-bromohexane we obtained Oa = 0.92±0.02 (unpublished).
This method was applied for the first time in 2012 [26], in which we averaged current and previous results of our group for four solvents: CCl4, acetone, ethanol and water. The ways to estimate coefficients a were not found at that time therefore it was suggested that a « 1 in all experiments. It was obtained using this assumption, DPIBF and Eq. 6 that absorbance of oxygen at 1270 nm in CCl4 is 1.5 times higher than we reported previously [21]. Besides, the relative values of the molar absorption coefficients for this band were proved to be proportional to the radiative rate constants (kr) of singlet oxygen also in water and ethanol. Deviation from the proportionality in water and alcohol reported in the preceding papers [22,23,25] was shown to be due to the strong increase of k„x for DPIBF in these media compared to organic solvents having no OH-groups (see [26,28] for details).
Papers of 2003-2012 solved principal experimental problems arising upon measurement of the absorption coefficients for dissolved oxygen. As a result, estimation of the numerical values for these coefficients in different environment was carried out [24-26]. As knowledge of the oxygen absorption coefficients is of fundamental importance for oxygen photonics, subsequent experiments were aimed at increasing the accuracy of our measurements.
For this goal, diode lasers having relatively narrow spectral band (4-6 nm) were employed. Coefficients a were calculated from the action spectra of trap oxygenation (Fig. 2) and spectra of laser radiation (in energy units) using Eq. 2 (see [27,28] for details). Two methods of analysis were applied. According to one of
them, ^1273 was carefully measured in CCl4 using Eq. 6 and relative rates of oxygenation of three traps -DPIBF, tetracene and rubrene upon photosensitized and direct oxygen excitation [28]. It was established that for all traps the relative oxygenation rates upon photosensitized and direct oxygen excitation normalized to power of exciting radiation and the fraction of light absorbed by porphyrins were ~104:
( Vpsr / Vlasr)/ (Ipsex/ ahas) «104. (7)
Hence, if 100% exciting light is absorbed by porphyrins, the efficiency of photosensitized oxygen excitation is 104 times higher than the efficiency of direct oxygen excitation. In real experiments porphyrin absorbed not more that 10% exciting light, therefore relative efficiency of photosensitized oxygen excitation was by one order smaller, therefore the ratio corresponding to Eq. 7 is about 103 [28].
In addition, it was found that ^1273 in CCl4 is almost two times greater than the value reported in ref. [21] (Table 1). The difference was shown to be mainly due to the fact that the real value of the molar absorption coefficient for tetracene was almost two times smaller than the literature value applied in the previous work [21] (see [28] for details). The obtained ^1273 in CCl4 was then used for correction of numerous results of our previous measurements of ^1273 in other organic solvents, which were estimated using Eq. 4 and CCl4 as the reference solvent [28] (Table 1). As mentioned above, these results were obtained upon direct laser excitation of oxygen from relative rates of DPIBF oxygenation in CCl4 and other organic solvents having no OH groups in their molecules.
According to the second method, the rates of DPIBF oxygenation were compared upon direct and photosensitized oxygen excitation in each solvent. The
Table 2 Correlation between obtained £1273 and the radiative rate constants (kr, s-1) in several solvents.
Parameters CCl4 C6F6 n-heptane Benzene Toluene Acetone Ethanol H2O
£1273*103, M-1cm-1 5.1 5.1 3.3 6.2 6.7 2.8 2.9 1.5
£1273, rel. units 1 1.0 0.65 1.2 1.3 0.55 0.57 0.29
kr/n2, rel. units [23,33] 1 1.0 0.62 1.2 1.2 0.53 0.54 0.25
Table 3 The radiative rate constants (k) and the phosphorescence quantum yields (Or) for triplet-singlet transitions in oxygen molecules dissolved in carbon tetrachloride estimated from the experimentally obtained £1273 and £765 (Table 1) using Eq. 8.
Transitions v, cm-1 Av, cm-1 £maxx103 M"1cm"1 kr, s-1 kr, s-1 a Or
>Ag(0) ^ 3Eg(0) 7855 ~103 5.1 0.60 1.17; 0.25 0.018
>Eg+(0)^ 3Eg(0) 13072 ~130 0.71 0.22 0.40;1.55 2.9*10-7
"This column shows the experimentally measured kr, reported in papers cited in Ref. [26];
b0r = krT where zis the lifetime of singlet oxygen equal to ~ 30 ms for the 1Ag state [37] and 130 ns for the 1Eg+ state in the reagent grade CCl4 ([38,39] and refs therein), n is 1.46 in CCl4.
most recent results of our studies are shown in Table 1. As seen from the Table, both methods yield similar values of the absorption coefficients. Correctness of the coefficients £1273 presented in Table 1 is supported by apparent correlation of their relative values with the relative values of k/n2 for the 1Ag-state of singlet oxygen in different solvents (Table 2).
Thus, the data of Table 2 indicate that one and the same mechanism is responsible for the solvent dependence of both kr calculated from the quantum yield of singlet oxygen phosphorescence at 1270 nm and £1273 obtained by methods of laser photochemistry. Both coefficients are about 1000 times greater than those in rarefied gas (see [21] for refs). It follows from Minaev's theoretical considerations that due to spinorbit coupling in O2 molecules and perturbations caused by their collisions with solvents, the a:Ag ^ X3 Eg-transition which is purely magnetic in the rarefied gas becomes allowed as electric dipole in solutions [34,35].
Hence, one can estimate the absolute value of kr from the obtained 51273 using the equation for the electric dipole transitions (so called, Kravetz equation). The simplest form of this equation was provided by Strickler and Berg [36]:
kr = n2 2.88X10"9 V2AV £oxgl/g2,
(8)
where n is the solvent refractive index, v is the transition frequency in cm-1, Av is the half-width of the oxygen absorption band (FWHM), £ox is the molar absorption coefficient of oxygen in M-1cm-1 and g1 and g2 are multiplicity of the initial and final states, g^/g2 = 3/1 (see also [26] and refs therein). Table 3 shows the results of application of Eq. 8 to the parameters of oxygen obtained in CCl4 (Table 1) and compares these results with the experimentally measured values obtained by different groups. Taking into account an
approximate character of Eq. 8 and scattering of the experimental numbers, correlation between the calculated and measured kr is satisfactory.
2.4 Absorbance at 765 nm
The first paper, in which A 765 was estimated in air-saturated carbon tetrachloride was published in 2007 [24]. Tetracene was used as the singlet oxygen trap and the tunable titan-sapphire laser was applied for oxygen excitation. The maximum of the photooxygenation action spectrum was observed at 765 nm (Fig. 3); A765 was roughly estimated to be 3.5 times smaller than A1273 and £765 ~10-3 M-1cm-1 was obtained [24]. Later, similar measurements were performed using DPIBF as a singlet oxygen trap. Values of A765 were estimated in carbon tetrachloride, acetone, ethanol and water. It was established that £765 ~10-3 M-1cm-1 in these solvents [26]. After further improvement of the measurement procedure and data analyses with accounting for coefficients a (see previous section) it was found that £1273 is almost two times greater than that obtained previously and the ratio (£1273^765) was equal to ~7 in CCl4. A slight increase of £765 with the increase of solvent polarity was also reported [27,28].
The most recent studies were performed based on Eq. 6 and comparison of the rates of DPIBF oxygenation upon direct and photosensitized oxygen excitation. In these experiments we used a new diode laser with the emission maximum at exactly 765 nm and
2.5 nm bandwidth. Several additional solvents were studied. Some results are presented in Table 1 and Fig. 5. It is seen that the ratio (£765/^273.) varies within 0.10.15 in non polar solvents, increases with the increase of solvent polarity to about 0.3 in acetone and ethanol and to 0.75 in water. Exclusion is 1-bromohexane, which despite low polarity, shows relatively high £765. This
effect can be attributed to the presence of heavy atom (bromine), which is known to enhance the 1Eg+(v=0) ^ 3Zg-(v=0) transition [9,31]. Based on these values of kr, the quantum yield of 1O2 phosphorescence at 765 nm can be estimated. Table 3 indicate that even in CCl4 the phosphorescence yield of this emission is very low. In other organic solvents and water the lifetime of 1Eg+ is known to be much smaller. For instance, it was estimated that ts « 30 ps in ethanol and 6.5 ps in water [38,39]. Therefore, the quantum yield of phosphorescence in these solvents should be by ~4 orders smaller than in CCl4, being about 10-11.
Fig. 5 Molar absorption coefficients corresponding to the main absorption maxima of molecular oxygen in several solvents.
2.5 Absorbance at 1070 nm
The absorption coefficient for the band at 1073 nm corresponding to the transition 1Ag (v=1) ^ 3Eg-(v=0) was reported in Refs. [26,28], in which oxygenation of tetracene and DPIBF solutions were studied under irradiation by the Nd-Yag laser (1064 nm) or by powerful diode lasers (1060 nm). As a result, we arrived to the ratio A1273/A1073 = 60±15. Fig. 6 illustrates relative intensities of the major oxygen absorption bands in CCI4.
Fig. 6 Relative values of the molar absorption coefficients in the maxima of the absorption spectrum of oxygen dissolved in CCl4 under normal conditions.
Comparison with Fig. 2 shows that the ratio A1073/A1273 obtained in our experiments is 30-40 times smaller than in the absorption spectra of oxygen dimols (O2)2 (see
also [9,11,26]). Judging by the ratio A1073/A1273, the concentration of oxygen dimols is very low under normal conditions being less than 3%. However, the ratio A1073/A1273 is a mirror image of the intensities of the major bands in the phosphorescence spectrum of monomeric singlet oxygen at 1274 and 1590 nm I1590/I1274 (see refs in [26,28]).
3 Conclusions
Thus, application of the methods of laser photochemistry allowed measurement of the absorption coefficients for major absorption bands of monomeric oxygen molecules dissolved in organic solvents and water under ambient conditions. This information has never been reported before our studies. Knowledge of these coefficients is obviously of basic importance for photonics of triplet and singlet oxygen because it provides information on the electronic structure of oxygen molecules and their interaction with environment. Recently, major results of our studies were confirmed by other researchers [29-31]. Although at this state of research numerical values reported by different groups sometimes do not quite coincide (only in acetone identical values were obtained by all authors) the reported values are still of the same order of magnitude. The difference does not exceed the factor of 3. As mentioned in the present paper and previous papers of our group, the values reported by our group also passed through certain evolution due to improvement of measurement techniques and data processing. As mentioned above, to obtain correct numbers one should take into account many parameters, determination of which is not always simple.
Biomedical importance of this research is also apparent. Our experiments demonstrate that direct laser excitation of oxygen molecules is a real natural process, which can be detected using modern laser and spectroscopic techniques. However, the effectiveness of this process is very low, because the absorption coefficients of oxygen have a very small value. The rate of photosensitized singlet oxygen production is 103-104 times greater than the rate of direct oxygen excitation. Therefore, it is difficult to expect that under moderate laser power allowed for photodynamic and laser therapy direct excitation of free oxygen dissolved in cell structures causes appreciable destruction of living tissues. It was shown recently that reasonable rates of cell damage can be observed under laser power of 200 W/cm2 that greatly exceeds the excitation power allowed for medical applications (usually < 200 mW/cm2) [40]. However, low-power laser radiation might influence enzyme-bound oxygen molecules whose concentration is much higher. The data are reported that singlet oxygen produces signaling effect, triggers expression of antistress genes and apoptosis and stimulates immune system response [4144]. In conclusion it is worth noting that according to our results, the lasers with the wavelength 765 nm are probably more appropriate for oxygen excitation in biomedical systems than lasers emitting at 1273 nm.
Indeed, both lasers have rather similar efficiency. However, the dark red light penetrates deeper and causes less heating (see [28] for more details).
Acknowledgements
We thank the Russian Foundation for Basic Research (project No 15-04-05500) and the program "Basic science for medicine" of the Russian Academy of Science for financial support. A.V. Lukovkin, director of the "Surgical innovative technologies Ltd", Moscow is gratefully acknowledged for diode lasers and technical assistance.
