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Photo-stability and photo-sensitizing characterization of selected sunscreens’ ingredients
Journal of Photochemistry and Photobiology A: Chemistry 332 (2017) 241–250
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Journal of Photochemistry and Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem
Photo-stability and photo-sensitizing characterization of selected sunscreens’ ingredients skib,c , Abdul Rahman Abida,b,* , Bronisław Marciniakb , Tomasz Pe˛dzin a Muhammad Shahid a b c
School of Chemical & Materials Engineering, National University of Sciences & Technology, Islamabad, Pakistan , Poland Faculty of Chemistry, Adam Mickiewicz University, Poznan Center for Advanced Technologies, Adam Mickiewicz University, Poznan, Poland
A R T I C L E I N F O
Article history: Received 11 March 2016 Received in revised form 25 July 2016 Accepted 31 August 2016 Available online 1 September 2016
A B S T R A C T
Sunscreen products are mostly used to protect human skin from adverse effects of ultra-violet light like DNA damage, photo-aging and sunburns. Photo-stability is one of the critical requirements for an effective sunscreen. However, most commercially available sunscreen compounds exhibit photoreactions leading to formation of harmful products. The objective of the current research was to investigate the photo-chemical and photo-physical properties of few selected sunscreens formulations and also to explore photo-stability and photo-sensitizing properties of few selected organic UV filters used in the sunscreens. Photo-degradation mechanisms of the selected sunscreens ingredients were analysed using time-resolved Laser Flash Photolysis and steady-state HPLC coupled with Mass Spectrometry. Oxybenzone (OXB) was revealed to be a sufficiently stable UV filter even after 24 h of radiation exposure, whereas, Avobenzone (AVOB) and Ecamsule (ECAM) were found to be a photounstable sunscreen. ã 2016 Published by Elsevier B.V.
1. Introduction The basic purpose of sunscreens is to protect the skin from the adverse effects like DNA damage, photo aging and sunburn due to ultraviolet (UV) radiations. Continuous exposure of our body parts to UV-light can cause significant damage to the skin [1]. UV-light can be divided in four sub brackets: UVC (100–290 nm), UVB (290– 320 nm), UVA1 (320–340 nm) and UVA2 (340–400 nm) [1]. UVClight does not reach the earth surface because that is largely absorbed by the atmosphere, whereas, UVB and UVA lights travel to the earth and cause undesirable effects on human skin [2]. Therefore, sunscreen filters are designed to either absorb or reflect the UVB and UVA categories. It has been documented that UVB can cause damage to the upper surface of the skin and does not penetrate into the skin; hence sunburn or DNA damage do not occur [4]. UVA-light penetrates deeper into the skin inside the DNA molecules and cause their photosensitized oxidation. This process takes place by two mechanisms: type ‘A’ and ‘B’. Type ‘A’ proceeds by yielding of
* Corresponding author at: School of Chemical & Materials Engineering, National University of Sciences & Technology, Islamabad, Pakistan. E-mail address: [email protected] (A.R. Abid). http://dx.doi.org/10.1016/j.jphotochem.2016.08.036 1010-6030/ã 2016 Published by Elsevier B.V.
radical ion in an excited state by electron or hydrogen atom transfer, and type ‘B’ activates formation of singlet molecular oxygen when excited sensitizer reacts with oxygen [3]. UV-light filters are categorized in two types, chemical and physical filters; the authors have worked on chemicals filters only. The chemical filters are further sub-divided in UVA filters UVB filters and broad spectrum filters which can absorb both types [4]. The sunscreens can be classified in three types of the chemicals organic UV-filters based on their working mechanisms. 1. Ultrafast excited state intramolecular proton transfer (ESIPT) based UV-filters like oxybenzone. 2. Filters requiring photo stabilizers like avobenzone. 3. Reversible Photo isomerization based UV-filters like ecamsule and/or mexoryl SX. Three ingredients of commercially available sunscreens (1) oxybenzone (2-hydroxy-4-methoxybenzophenoe), (2) avobenzone (1-(4-Methoxyphenyl)-3-(4-tert-butylphenyl) propane-1,3dione) and (3) Ecamsule (Sulfonic Acid based) were characterized for their photo-stability and photosensitizing properties (Fig. 1). Table 1 [5] shows properties of the selected UV filters:
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2.2.2. Fluorescence spectrometry Singlet oxygen measurement was performed on FluoTime 300 fluorescence spectrophotometer (Pico-Quant). The experiment was performed at excitation wavelength 310 nm in 1 cm of quartz cell.
Fig. 1. Chemical structures of various sunscreen formulations: (a) Oxybenzone (OXB); (b) Avobenzone (AVOB); (c) Ecamsule (ECAM).
2.2.3. Absorption spectroscopy Femtosecond transient absorption spectra and kinetics were performed on a commercial setup from ultrafast systems (USA) and Spectra-Physics (USA). The setup allowed measurement of events as short as 200 fs. The pump’s pulse energy was fixed to 5 mJ. The absorbance was set to about 0.5 at the excitation wavelength of 355 nm in a 2 mm quartz cell. The transient absorption kinetics’ traces were analysed using commercial software ‘Surface Xplorer 2.2’ developed by Ultrafast Systems. All transient absorption experiments were performed at room temperature. Nanosecond laser flash photolysis was used to measure transient absorbance; the pump’s pulse energy was kept in the range 30–40 mJ and the absorbance was fixed to 0.5 at excitation wavelengths of 355 nm and 266 nm in 1 cm quartz cell.
Table 2 summarizes materials used in experimentation for characterization. It highlights materials investigated along with the helping materials for investigation.
2.2.4. Chromatography HPLC (high performance liquid chromatography) was performed using a commercial machine Ultimate 3000 (Thermo Scientific) with a C4 column. All experiments were performed by UV/Vis detector at the detector wavelengths 190 nm and 289 nm). The sample was irradiated at 355 nm wavelength using monochromatic light; the power of a laser was fixed at 80 mW (continuous irradiation).
2.2. Characterization techniques
3. Results and discussion
2.2.1. UV–vis spectroscopy Steady-state absorption spectra were measured by using a Cary100 UV/Vis spectrophotometer (Agilent). The absorption spectra was obtained from 200 nm to 500 nm.
3.1. Oxybenzone (OXB)
2. Experimental 2.1. Materials used for experimentation
It is evident from Fig. 2 that OXB absorbs light in UVA1 and UVB regions; the overall wavelength covered by OXB absorption ranges
Table 1 Documented Properties of UV filters Selected for Study. UV-filter
Range Max. conc. used
Function
Precautions/Comments
Oxybenzone UVB UVA2
6%
Absorbs UVA rays
Photostable Helps stabilize other UV filters Absorbed by skin;
Avobenzone UVA
3%
Absorbs full spectrum of UVA rays
Mexoryl SX/ Ecamsule
3%
Absorbs UV rays, then releases the UV rays Photo-stable as thermal energy; No skin penetration Water-soluble Does not protect against entire UV spectrum; needs to be combined with other filters for good protection
UVA1 UVA2
Extremely photo-unstable Degrades in light Oil-soluble Tends to be unstable in the presence of octinoxate Can be stabilized by photo-stablizer
Table 2 Materials used for experimentation and characterization. Sr.#
Material
Source/specs
1 2 3 4
Acetonitrile (AcN) solvent Oxybenzone (OXB), Avobenzone (AVOB) and Ecamsule (ECAM) Purified water Argon gas
Reagent grade; Sigma Aldrich Sigma Aldrich Millipore (Milli-Q) High purity
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Fig. 2. Absorption Spectrum of Oxybenzone (OXB) with absorption of 0.3 at 355 nm of wavelength and concentration in 20 mM. Absorption maximum was 0.84 at 287 nm.
between 290 nm and 360 nm. Therefore, OXB is one of the most common UV absorbers used in the sunscreen. The absorption spectrum of OXB shows the three maxima’s of absorption band shifts at: 1st ! 241 nm; 2nd ! 288 nm and 3rd ! 324 nm. The 1st and 2nd absorption bands are believed to be due to p ! p* because of the two benzene rings presents in molecular structure of OXB, whereas, the 3rd band is presumably due to the n ! p* because of the carboxyl group present in OXB molecule [6]. The sample of OXB and Met (Methionine) was continuously exposed to radiation for 3h and the exposed and unexposed samples were injected in HPLC for characterization. Fig. 3 reveals that before irradiation the chromatogram exhibited two peaks; the peak at retention time 0.8 min generated from Met, whereas, the second peak at retention time 1.25 min was produced from OXB. As
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evident from Fig. 3, no noticeable difference appeared between exposed and unexposed spectra. Is was evident that OXB proved to be highly photo-stable even in the presence of Met. Careful analysis of the HPLC chromatogram reveals a small peak at retention time 1.65 min and very small speak after the OXB peak not too much visible in the chromatogram. OXB is based on ultrafast excited state intermolecular proton transfer reaction (ESIPT) [7] and does not efficiently yield any stable photoproducts; this makes the OXB significantly photo-stable even after a prolong time of irradiation by UV-light. In ESIPT based reaction, enol form of OXB absorbs energy and then transforms into an excited state, before reverting back to the ground state by releasing its extra energy as keto emission [8]; the mechanism in shown in Fig. 4. The Figure reveals that the molecule absorbs energy to transform into singlet excited state from its ground state followed by transfer of its energy into an excitedstate molecule. The excited state is immediately quenched to ground state by internal conversion transferring back the energy of the molecule back to the OXB at ground state. OXB proved to be highly photo-stable against steady-state irradiations even at prolonged exposure for 24h at high photon flux of 80 mW, in the presence of Met; it was decided to test the system for possible transient product formation. For this investigation, nLFP was used with an excitation at 355 nm. When a high laser pulse energy was used (ca. 30–40 mJ/pulse), a rather weak transient absorption was observed being maximum at 390 nm (Fig. 5). The OXB molecule was excited by a pump pulse of 355 nm and the kinetic of the excited molecule was measured at 380 nm of probe laser as shown in Fig. 5; the figure shows kinetic trace of the OXB. The kinetic trace explains that after the excitation transient absorption decays to its initial value in a microsecond. There was no difference between the kinetic traces, in the argon saturated and oxygen saturated samples for OXB In order to observe some transient in the nLFP experiment, methionine was added as a potential scavenger of OXB-derived
Fig. 3. HPLC chromatogram of a mixture of OXB in the presence of Met (OXB 1mM + Met 10 nM) before [(black line monitored at 190 nm) and (blue line monitored at 289 nm),] and after [(red line monitored at 190 nm) and (maroon line monitored at 289 nm)]. In set the chromatogram is 2D for more clear identification of compounds present in the solution. The flow rate was 0.800 mL/min and injection volume was 20 mL. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Keto-enol tautomerization of OXB (R1 = OCH3, R2 = H) (a) and simplified ESIPT reaction mechanism (b).
transients (Fig. 5). However, after measuring the nLFP of sample in the presence of scavenger at the same experimental parameters and after normalization of the traces, no scavenging effect of the Met was observed on OXB; the decay remained approximately unchanged. That verified that OXB was highly photo-stable even in the presence of Met as quencher compound with a very less photoliability. 3.2. Avobenzone (AvoB) The UV/Vis Absorption spectroscopy of Avobenzone shows two major peaks (Fig. 6); the absorption band exhibiting maximum absorption at around 350 nm can be assigned to the enol form of AVOB while the weaker band appearing at 270 nm can be attributed to diketo form of AVOB [9]. It is evident from Fig. 6 that before exposure of the AVOB to UV light the enol form appeared to be dominant compared with diketo. Fig. 6 further reveals that the absorption band of enol decreased and that of diketo increased with exposure time. The constitutional isomer enol and diketo readily interconvert upon exposure to the photons by a chemical reaction called ‘tautomerization’. Hence, the enol and diketo can be termed as tautomer of each other in the case of AVOB. This enol to diketo migration within the molecule can be attributed to proton transfer to the adjacent double bond [10]. Various isomers show various absorption bands as the transitions of bonding orbitals to anti-bonding orbitals vary accordingly. For example, p ! p* transitions are preferable over
Fig. 5. nLFP of Oxybenzone (OXB) at 355 nm excitation wavelength without quencher (black, argon saturated solution) and with quencher added (red, 10 mM of Met) after normalization. Here, Methionine (Met) acts as a quencher. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
s ! s* since the former transitions requires lesser energy photons compared to the later. UV/Vis Absorption spectra of oxygen-saturated and argonsaturated AVOB when irradiated up to 2h, appear to be similar as shown in Fig. 6. However, prolonged exposure to UV light up to 3h generated a significant difference in the absorption bands of the AVOB both under oxygen and argon saturated conditions compared with unexposed condition (Fig. 7). That clear indication that the photoreactions of both cases were different for oxygen and argon saturated samples. Knowing the photo-unstability of AVOB, HPLC apparatus was employed in order to investigate the photoproducts generated as a result of UV irradiation. Fig. 8 reveals HPLC chromatograms of AVOB samples before and after 3h irradiation; comparison between immediately irradiated and after one day of irradiation is also demonstrated. It is evident that before irradiation only two peaks existed in the chromatogram; the higher intensity peak at retention time 1.5 min is believed to be from enol and the lower intensity peak at retention time 0.75 min is attributed to diketo. However, soon after irradiation, the enol peak intensity is decreased, whereas, intensity of diketo peak is increased. Furthermore, new peaks were observed in early retention times between 0.20 min to 0.60 min; these peaks may be evolved due to expected photo-products of AVOB. The 3h irradiated sample stored for one day revealed a changed chromatogram. The enol and diketo peaks appeared to be reversed and photo-products became stale; the enol, however, was not fully reversible as the increase was not exactly equal to the peak intensity before irradiation. This decrease in the intensity of the enol peak was expected because of stable photo-product observed in the early retention time of chromatogram. Fig. 9 displays the HPLC chromatograms of the argonsaturated samples of AVOB before and after irradiation of UVlight for 3h. Argon-saturated sample of AVOB followed similar trend as shown in Fig. 8. The enol peak decreased at retention time 1.5 min whereas the diketo peak increased at retention time 0.75 min after irradiation; few photoproducts were also observed at early retention times between 0.2–0.70 min in the chromatogram. Comparing Figs. 8 and 9, it is indicated that the photoproducts in case of argon-saturated were more compared with oxygen-saturated samples. Hence, it may be stated that degradation of AVOB in the presence of argon was more as compared to oxygen generating additional photo-products. Furthermore, in case of argon-saturated AVOB irradiated samples, the reconversion of the diketo into enol form of AVOB was not significant compared with oxygen-saturated samples; the photoproducts also appeared to be stable. Both argon-saturated and oxygensaturated solutions showed a distinct photodegradation, however, the products detected in HPLC chromatograms were noticeably different.
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Fig. 6. Absorption Specta of Avobenzone (AVOB) at various irradiation exposure (a) Oxygen-saturated (left) and (b) argon-saturated.
Fig. 7. Comparison of Absorption Spectrum of Avobenzone (AVOB) for Argon Saturated and Oxygen Saturated. Before and after irradiation at 355 nm of wavelength.
The photoproducts indicated at retention times 0.20–0.70 min in Figs. 8 and 9 for oxygen-saturated and argon-saturated samples, were collected with help of fraction collector in HPLC. The collected fractions were injected by syringe injector to LC–MS Mass Spectrometer for analytical analyses. The fraction from oxygen-saturated AVOB sample was revealed at 265.18 Da in m/z spectrum for the oxygen saturated samples the 1 fraction was collected and after injecting that fraction of LC–MS; it was the only photoproduct observed after irradiation in this case, as displayed in Fig. 10 (a). Fig. 10 (b,c) reveal m/z spectra of argon-saturated sample after irradiation; the number of peaks were more so two fractions were analysed to identify the photoproducts. One fraction displayed a dominant peak at retention time from 0.20 min to 0.50 min, at 265.18 Da similar to the oxygen-saturated sample, whereas, the second displayed a peak at 301.144 Da at retention time from 0.50 min to 0.80 min. These analyses indicated presence of two photoproducts in argon-saturated sample with molecular masses of 264.17 Da and 300.13 Da. The common photoproduct with molecular mass of 265.18 Da appearing in both oxygen- and argon-saturated samples, was
further analysed as shown in Fig. 11, however, the molecular structure could not be revealed. After establishing the photo-liability of AVOB (irradiated for 3h at high photon flux of 80 mW) by steady-state irradiation experimentation, further testing was performed on the system for possible transient product formation or triplet formation. Nanosecond laser flash photolysis (nLFP) of oxygen- and argonsaturated AVOB samples was determined at excitation wavelengths of 355 and 266 nm, as shown in Fig. 12. At excitation wavelength of 355 nm (excitation of enol), no transient absorption was observed, however, there was a bleaching (negative absorption) observed possibly due to depopulation of a ground state of AVOB. The negative signal was recovered within nanoseconds suggesting no triplet formation. The negative absorption also shows that the extinction coefficient at ground state was higher than the extinction coefficient of excited state so according to the Lambert-Beer law [11] the change in optical density should be negative. In both cases oxygen- and argon-saturated samples, the offset between the baseline and decay curve appeared because of formation of the photoproducts during excitation. Interestingly, in kinetics at excitation 355 nm (Fig. 12(a)), the argon-saturated sample showed a relatively fast decay in the beginning before coming down to a steady state approaching to the decay value of oxygen-saturated sample. This fast decay may be attributed to absorption of diketo form of AVOB. For further confirmation the system was tested for possible transient product formation or triplet formation at excitation wavelength 266 nm (Fig. 12(b)). The diketo form when excited at 266 nm, generated an intensive transient absorption signal as detected by nLFP and the lifetime of this transient was found to be dependent on the presence of oxygen. Therefore, that absorption was believed to be due to the triplet state of diketo AVOB formation. The Fig. 12(b) also shows an ultrafast triplet formation in diketo in argon-saturated sample at excitation wavelength 266 nm. Hence, it can be deduced from this experiment that AVOB usage for the sunscreens at lower wavelength are not safe. The photo-isomerization of compound was oxygen dependent occur through the triplet excited state and can be one of the rational explanation for limited stabilization of Avobenzone [12,13]. Fig. 13(a) shows transient spectrum of AVOB at 355 nm excitation wavelength; the spectrum shows that maximum light was absorbed 360 nm. The species absorbing at the wavelength of 320 nm were not significantly absorbed at the maximum depletion wavelength this depletion wavelength helped to estimate the
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Fig. 8. HPLC chromatogram of Oxygen saturated Avobenzone sample just after irradiation (a) and after one day same sample without irradiation (b). The chromatogram at 190 nm detector wavelength. The flow rate was 0.800 mL/min and injection volume is 20 mL for the analysis with mobile phase of 3:2 AcN:H2O.
Fig. 9. HPLC Chromatogram of Argon saturated Avobenzone sample just after irradiation (a) and after one day same sample without irradiation (b). The detector wavelength is 190 nm. The flow rate was 0.800 mL/min and injection volume was 20 mL for the analysis with mobile phase of 3:2 AcN:H2O.
extinction coefficient of enol rotamer form of AVOB. The extinction coefficient for rotamer was estimated about 19000 M1 cm1 10% with the help beer lambert law. As shown in Fig. 13(b) the transient spectrum of AVOB at 266 nm excitation wavelength, the maximum light was absorbed at 390 nm. It also reveals that diketo continues to absorb even at substantially longer probe wavelengths indicating a long-lived transient formation. The transient spectra from 440 to 550 nm also determines variation in optical density without approaching absolute zero. The triplet–triplet energy transfer mechanism was one of the origin of efficient triplet state population confirming the triplet state transient at 390 nm. Fig. 14 shows that singlet oxygen formation of AVOB at the excitation wavelength of 310 nm. The triplet formation was confirmed by an experiment performed in the presence of oxygen, where the singlet oxygen was observed at 1270 nm (Fig. 14 shows formation of the singlet oxygen from AVOB). The efficiency of this near-infra-red emission was found to be dependent on the oxygen
concentration as it disappeared for argon-saturated solutions. This leads to an important conclusion that formation of singlet Oxygen (through triplet–triplet energy transfer between 3AvoB* and 3O2) being a highly reactive species, may induce irreversible modifications. Singlet oxygen is an excited state of oxygen and highly energetic form of oxygen. Furthermore singlet oxygen has been found to be highly unstable and reactive radical towards organic compounds [14]. Singlet oxygen is commonly involved in damage to skin or tissues during the sunlight exposure. This can also be stated that the usage of AVOB as sunscreen above 320 nm of wavelength was safer. Further investigation on photo-reactivity of AVOB was performed using a femtosecond time resolved spectroscopy. Varying kinetics for different probe wavelengths at excitation wavelength of 355 nm are shown in Fig. 15. It is evident from the kinetics that life time for the excited species to ground state was about 40–50 ps. The remaining offset of absorption can be attributed to monophotonic ionization and deprotonation of the
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Fig. 10. LC–MS m/z Spectra for Oxygen Saturated (a) and Argon saturated samples (b) fraction for Oxygen-saturated and (c) fraction for argon-saturated samples.
AVOB molecule. This indicates that diketo form of AVOB remained excited for some time before going back to the enol stable form to the ground state. The offset in decaying can also be attributed to the photoproducts formed during excitation since all excited
Fig. 11. m/z spectra of fragmentation of 265.18 photoproduct present in both oxygen and argon saturated samples.
molecules cannot came back to their ground state and convert into stable photoproducts. 3.3. Ecamsule (ECAM) The UV/Vis absorption spectra of ECAM were determined before and after the irradiation at 355 nm using water and AcN as solvent in 2:3 ratio (Fig. 16). The ECAM before irradiation shows absorption band maximum at 350 nm with the molar absorption coefficient of 4.5 104 dm3mol1 cm1. The useful range for absorption of UV light is 300–400 nm which covers the UVA region for potential use for sunscreens. The UV/vis absorption spectra were measured after continuous irradiation for 2h at 355 nm of monochromatic UV light. As evident from Fig. 16, a significant decrease in the absorption band appeared due to photo degradation with an increase in time for irradiation, however, after 2h of irradiation no significant decrease was observed. It was also observed in Fig. 16 that the maximum of the absorption was also varying by increasing the time of irradiation. The maximum absorption appeared towards lower wavelength of 300 nm. This change in the maximum absorption also indicates formation of intermediates during degradation of the ECAM by photon excitation. It has been demonstrated [11] that the compounds which show photo induced isomerization when irradiated by UV light exhibit
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Fig. 12. Kinetics of Avobenzone (AVOB) at 355 nm (a) and at 266 nm (b) with argon and oxygen saturated. The number of averages taken during kinetic traces were 15. The solvent used AcN.
Fig. 13. Transient Absorption Spectrum of AVOB at excitation wavelength 355 nm (a) and at 266 nm (b) with different lifetime.
Fig. 14. Singlet Oxygen Formation excitation wavelength at 310 nm excitation wavelength; the spectrum was obtained from averages of 6 spectra.
interchange of isomers to one another when both the isomers have different levels of maximum absorption causing a displacement in the maximum absorption spectrum with an increase in the irradiation time. As discussed the ECAM works on the principle of reversible photo-isomerization, followed by photo excitation. ECAM absorbs UV light and releases energy in the form of heat without penetration into the skin. As the ECAM absorbs the photons of UV light molecule change from trans-isomer in to cis-isomer. The change from trans ! cis isomers being an ultrafast process, was determined using a femtosecond laser flash photolysis. Fig. 17 shows the kinetics traces of the ECAM molecule at excitation wavelength of 355 nm. It was observed that the kinetics yielded a quite short excited state (absorption maximum at 560 nm), presumably due to the initially excited singlet state with a life time of about 10 ps. The decay was substantially fast indicating that the molecules were going to their singlet excited state and coming back to ground state quickly in few picoseconds by internal conversion. In the presence
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Fig. 17. Kinetics of Ecamsule (ECAM) at Excitation wavelength of 355 nm. Kinetic trace probe wavelength 608 nm. ECAM was dissolved in AcN. Fig. 15. Femtosecond Kinetics of AVOB at various pulse wavelengths using an excitation wavelength of 355 nm. The kinetic traces were in three different probe wavelengths 450, 416 and 439 nm.
Fig. 16. UV/Vis Absorption Spectra of Ecamsule (ECAM) before and after irradiation. Before irradiation Absorbance was 0.47 at 355 nm with a maximum value of 0.61 at 340 nm. The irradiation was at 355 nm. The solvent was 3:2 AcN and H2O.
of AcN solvent ECAM showed a weak fluorescence [15] which also indicated that the molecule was going from ground state to first singlet excited state and then coming back to the ground state in few picoseconds. It decays quite fast probably due to the efficient photo-isomerization. On the other hand its photodegradation suggests that the most desired (in term of its application as a sunscreen) reversible photo-isomerization is not the only deactivation channel of its excited state. The femtosecond transient absorption spectrum of ECAM is shown in Fig. 18. The transient absorption of ECAM molecule displays absorbance of the probe pulse in the range of 500–650 nm. Interestingly displacement in the transient absorption was observed with variation in life times. The displacement in transient absorption spectrum is an evidence for vibrational cooling during depopulation of ECAM molecules at their ground states. The molecules were photoexcited with some excess vibrational energy in a solution before redistribution of energy took place at
Fig. 18. Transient Absorption Spectrum of Ecamsule (ECAM) in solvent AcN. The Excitation wavelength was 355 nm. The pulse energy was 5 mJ. The absorption was 0.5 in 2 mm of quartz cell.
vibrational manifold of the ground state. As the molecules relaxed to ground state of vibrational manifold, the energy was transferred to solvent present in excess amount releasing energy to solvent, by the process called vibrational cooling. The monitoring of the excess energy dissipation from solute to solvent play a vital role in photoinduced chemical reactions [16]. In the broader way, vibrational cooling should occur through a ladder process and by a multiphonon process. In the ladder process the vibration occur through lower order interactions with the neighbouring phonons in vibrational states of energy. The excess energy in the vibrational states fall the vibrational ladder step by step by losing the energy to phonons. In the multiphonon process, the excess energy was lost by the higher order interactions with a bunch of multiphonon states [17]. The vibrational cooling was ultrafast of the order of 10 ps. During the vibrational cooling it was quite possible that the solvent reorientation and solvent coordination (the coordination of molecule solvent H atom will change to coordination of solvent N atom) occur which was proportional to is vibrational cooling rate [18].
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4. Conclusions
Acknowlegement
Oxybenzone (OXB) proved to be a stable UV filter. Even after 24 h of irradiation there was no photodegradation of OXB even in the presence of Met as a potential quencher (except a small peak observed on a chromatogram after 24 h of irradiation). In the nLFP experiments in which Met was used as a quencher, there was no change in the kinetics of the transient decay. This suggests that although some transients are formed after OXB excitation (most likely identified as phenoxyl radicals), they are not very reactive and are not efficiently yielding any stable photoproducts. In general, the OXB sunscreen was shown to be photo-stable, however, even low concentrations of free radicals may lead to undesired adverse effects to the skin protein. AVOB appeared to be rather unstable UV sunscreen and it photodegrades with time of irradiation. However, it is a most commonly used chemical sunscreen and it is usually used in a combination with other molecules acting as AVOB photostabilizers. The post-irradiation HPLC analysis showed few prominent photoproducts on the chromatogram and also exhibited changes from enol to diketo form of AVOB. A diketo form of AVOB absorbs light at <300 nm, whereas, the enol form shows absorption at around 320–360 nm range. When the 355 nm irradiations were carried out (exciting exclusively the enol form) a prominent and irreversible photodegradation of AVOB was observed. Another reaction was a reversible phototautomarization, where the enol form was converted to the diketo form. However, a changed phenomenology was observed when the diketo form was excited at 266 nm. The intensive transient absorption signal was detected in nLFP and the lifetime of this transient was found to be dependent on the presence of oxygen. Therefore we could conclude that this absorption was due to the triplet state of diketo AVOB formation. The efficiency of the nearinfra-red emission was found to be dependent on the oxygen concentration as it disappeared completely for argon-saturated solutions. Formation of singlet oxygen may induce irreversible modifications e.g. in skin protein, leading to tissue photodegradation and other harmful effects to the skin cells. Ecamsule (ECAM) was not found to be a photo-stable sunscreen as it photodegraded with irradiation by UV light as found by the absorption spectroscopy. It decays quite fast due to the efficient photoisomerization. On the other hand, its photodegradation suggests that the most desired (in terms of its application as a sunscreen) reversible photoisomerization is not the only deactivation channel of its excited state. The other processes (triplet formation, bond breakages) remain unknown and will require further investigation. This chemical sunscreen should also be used with photo-stabilizers, otherwise it will photodegrade quickly.
I truly acknowledge the support of Erasmus Mundus Master SERP-Chem, especially Prof. Bohdan Skalski and Prof. Sandrine LACOMBE. I also thank to all my colleagues in Laboratory of Chemical Physics, Faculty of Chemistry UAM and providing me all the facilities during my research. References [1] J. Kockler, M. Oelgemöller, S. Robertson, B.D. Glass, Photostability of sunscreens, J. Photochem. Photobiol. C Photochem. Rev. 13 (2012) 91–110. [2] S. Madronich, R.L. McKenzie, L.O. Björn, M.M. Caldwell, Changes in biologically active ultraviolet radiation reaching the Earth’s surface, J. Photochem. Photobiol. B Biol. 46 (1998) 5–19. [3] M.V.R. Velasco, Broad spectrum bioactive sunscreens, Int. J. Pharm. 363 (2008) 50–57. [4] P.U. Giacomoni, L. Teta, L. Najdek, Sunscreens: the impervious path from theory to practice, Photochem. Photobiol. Sci. 9 (2010) 524–529. [5] UV Filters Chart: Sunscreen Active Ingredients | Skinacea.com. at. [6] L.A. Baker, M.D. Horbury, S.E. Greenough, M.N.R. Ashfold, V.G. Stavros, Broadband ultrafast photoprotection by oxybenzone across the UVB and UVC spectral regions, Photochem. Photobiol. Sci. 14 (2015) 1814–1820. [7] M.T. Ignasiak, C. Houée-Levin, G. Kciuk, B. Marciniak, T. Pedzinski, A reevaluation of the photolytic properties of 2-hydroxybenzophenone-based UV sunscreens: are chemical sunscreens inoffensive? Chemphyschem 16 (2015) 628–633. [8] J. Zhao, S. Ji, Y. Chen, H. Guo, P. Yang, Excited state intramolecular proton transfer (ESIPT): from principal photophysics to the development of new chromophores and applications in fluorescent molecular probes and luminescent materials, Phys. Chem. Chem. Phys. 14 (2012) 8803–8817. [9] G.J. Mturi, B.S. Martincigh, Photostability of the sunscreening agent 4-tertbutyl-40 -methoxydibenzoylmethane (avobenzone) in solvents of different polarity and proticity, J. Photochem. Photobiol. A Chem. 200 (2008) 410–420. [10] C. Paris, V. Lhiaubet-Vallet, O. Jiménez, C. Trullas, M.A. Miranda, A blocked diketo form of avobenzone: photostability, photosensitizing properties and triplet quenching by a triazine-derived UVB-filter, Photochem. Photobiol. 85 (2016) 178–184. [11] R. Berera, R. van Grondelle, J.T.M. Kennis, Ultrafast transient absorption spectroscopy: principles and application to photosynthetic systems, Photosynth. Res. 101 (2009) 105–118. [12] V. Lhiaubet-Vallet, Filter-filter interactions. Photostabilization, triplet quenching and reactivity with singlet oxygen, Photochem. Photobiol. Sci. 9 (2010) 552–558. [13] S. Afonso, Photodegradation of avobenzone: stabilization effect of antioxidants, J. Photochem. Photobiol. B 140 (2014) 36–40. [14] M.C. DeRosa, R.J. Crutchley, Photosensitized singlet oxygen and its applications, Coord. Chem. Rev. 233 (2002) 351–371. [15] A. Cantrell, D.J. McGarvey, L. Mulroy, T.G. Truscott, Laser flash photolysis studies of the UVA sunscreen mexoryl SX, Photochem. Photobiol. 70 (1999) 292–297. [16] Structure, Fluctuation, and Relaxation in Solutions, Elsevier, 1995 at . [17] S.H. Lin, A.A. Villaeys, Y. Fujimura, Advances in Multi-Photon Processes and Spectroscopy, World Scientific, 1996 at . [18] J.D. Simon, Ultrafast Dynamics of Chemical Systems, Springer Science & Business Media, 2012 at .
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem
Photo-stability and photo-sensitizing characterization of selected sunscreens’ ingredients skib,c , Abdul Rahman Abida,b,* , Bronisław Marciniakb , Tomasz Pe˛dzin a Muhammad Shahid a b c
School of Chemical & Materials Engineering, National University of Sciences & Technology, Islamabad, Pakistan , Poland Faculty of Chemistry, Adam Mickiewicz University, Poznan Center for Advanced Technologies, Adam Mickiewicz University, Poznan, Poland
A R T I C L E I N F O
Article history: Received 11 March 2016 Received in revised form 25 July 2016 Accepted 31 August 2016 Available online 1 September 2016
A B S T R A C T
Sunscreen products are mostly used to protect human skin from adverse effects of ultra-violet light like DNA damage, photo-aging and sunburns. Photo-stability is one of the critical requirements for an effective sunscreen. However, most commercially available sunscreen compounds exhibit photoreactions leading to formation of harmful products. The objective of the current research was to investigate the photo-chemical and photo-physical properties of few selected sunscreens formulations and also to explore photo-stability and photo-sensitizing properties of few selected organic UV filters used in the sunscreens. Photo-degradation mechanisms of the selected sunscreens ingredients were analysed using time-resolved Laser Flash Photolysis and steady-state HPLC coupled with Mass Spectrometry. Oxybenzone (OXB) was revealed to be a sufficiently stable UV filter even after 24 h of radiation exposure, whereas, Avobenzone (AVOB) and Ecamsule (ECAM) were found to be a photounstable sunscreen. ã 2016 Published by Elsevier B.V.
1. Introduction The basic purpose of sunscreens is to protect the skin from the adverse effects like DNA damage, photo aging and sunburn due to ultraviolet (UV) radiations. Continuous exposure of our body parts to UV-light can cause significant damage to the skin [1]. UV-light can be divided in four sub brackets: UVC (100–290 nm), UVB (290– 320 nm), UVA1 (320–340 nm) and UVA2 (340–400 nm) [1]. UVClight does not reach the earth surface because that is largely absorbed by the atmosphere, whereas, UVB and UVA lights travel to the earth and cause undesirable effects on human skin [2]. Therefore, sunscreen filters are designed to either absorb or reflect the UVB and UVA categories. It has been documented that UVB can cause damage to the upper surface of the skin and does not penetrate into the skin; hence sunburn or DNA damage do not occur [4]. UVA-light penetrates deeper into the skin inside the DNA molecules and cause their photosensitized oxidation. This process takes place by two mechanisms: type ‘A’ and ‘B’. Type ‘A’ proceeds by yielding of
* Corresponding author at: School of Chemical & Materials Engineering, National University of Sciences & Technology, Islamabad, Pakistan. E-mail address: [email protected] (A.R. Abid). http://dx.doi.org/10.1016/j.jphotochem.2016.08.036 1010-6030/ã 2016 Published by Elsevier B.V.
radical ion in an excited state by electron or hydrogen atom transfer, and type ‘B’ activates formation of singlet molecular oxygen when excited sensitizer reacts with oxygen [3]. UV-light filters are categorized in two types, chemical and physical filters; the authors have worked on chemicals filters only. The chemical filters are further sub-divided in UVA filters UVB filters and broad spectrum filters which can absorb both types [4]. The sunscreens can be classified in three types of the chemicals organic UV-filters based on their working mechanisms. 1. Ultrafast excited state intramolecular proton transfer (ESIPT) based UV-filters like oxybenzone. 2. Filters requiring photo stabilizers like avobenzone. 3. Reversible Photo isomerization based UV-filters like ecamsule and/or mexoryl SX. Three ingredients of commercially available sunscreens (1) oxybenzone (2-hydroxy-4-methoxybenzophenoe), (2) avobenzone (1-(4-Methoxyphenyl)-3-(4-tert-butylphenyl) propane-1,3dione) and (3) Ecamsule (Sulfonic Acid based) were characterized for their photo-stability and photosensitizing properties (Fig. 1). Table 1 [5] shows properties of the selected UV filters:
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2.2.2. Fluorescence spectrometry Singlet oxygen measurement was performed on FluoTime 300 fluorescence spectrophotometer (Pico-Quant). The experiment was performed at excitation wavelength 310 nm in 1 cm of quartz cell.
Fig. 1. Chemical structures of various sunscreen formulations: (a) Oxybenzone (OXB); (b) Avobenzone (AVOB); (c) Ecamsule (ECAM).
2.2.3. Absorption spectroscopy Femtosecond transient absorption spectra and kinetics were performed on a commercial setup from ultrafast systems (USA) and Spectra-Physics (USA). The setup allowed measurement of events as short as 200 fs. The pump’s pulse energy was fixed to 5 mJ. The absorbance was set to about 0.5 at the excitation wavelength of 355 nm in a 2 mm quartz cell. The transient absorption kinetics’ traces were analysed using commercial software ‘Surface Xplorer 2.2’ developed by Ultrafast Systems. All transient absorption experiments were performed at room temperature. Nanosecond laser flash photolysis was used to measure transient absorbance; the pump’s pulse energy was kept in the range 30–40 mJ and the absorbance was fixed to 0.5 at excitation wavelengths of 355 nm and 266 nm in 1 cm quartz cell.
Table 2 summarizes materials used in experimentation for characterization. It highlights materials investigated along with the helping materials for investigation.
2.2.4. Chromatography HPLC (high performance liquid chromatography) was performed using a commercial machine Ultimate 3000 (Thermo Scientific) with a C4 column. All experiments were performed by UV/Vis detector at the detector wavelengths 190 nm and 289 nm). The sample was irradiated at 355 nm wavelength using monochromatic light; the power of a laser was fixed at 80 mW (continuous irradiation).
2.2. Characterization techniques
3. Results and discussion
2.2.1. UV–vis spectroscopy Steady-state absorption spectra were measured by using a Cary100 UV/Vis spectrophotometer (Agilent). The absorption spectra was obtained from 200 nm to 500 nm.
3.1. Oxybenzone (OXB)
2. Experimental 2.1. Materials used for experimentation
It is evident from Fig. 2 that OXB absorbs light in UVA1 and UVB regions; the overall wavelength covered by OXB absorption ranges
Table 1 Documented Properties of UV filters Selected for Study. UV-filter
Range Max. conc. used
Function
Precautions/Comments
Oxybenzone UVB UVA2
6%
Absorbs UVA rays
Photostable Helps stabilize other UV filters Absorbed by skin;
Avobenzone UVA
3%
Absorbs full spectrum of UVA rays
Mexoryl SX/ Ecamsule
3%
Absorbs UV rays, then releases the UV rays Photo-stable as thermal energy; No skin penetration Water-soluble Does not protect against entire UV spectrum; needs to be combined with other filters for good protection
UVA1 UVA2
Extremely photo-unstable Degrades in light Oil-soluble Tends to be unstable in the presence of octinoxate Can be stabilized by photo-stablizer
Table 2 Materials used for experimentation and characterization. Sr.#
Material
Source/specs
1 2 3 4
Acetonitrile (AcN) solvent Oxybenzone (OXB), Avobenzone (AVOB) and Ecamsule (ECAM) Purified water Argon gas
Reagent grade; Sigma Aldrich Sigma Aldrich Millipore (Milli-Q) High purity
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Fig. 2. Absorption Spectrum of Oxybenzone (OXB) with absorption of 0.3 at 355 nm of wavelength and concentration in 20 mM. Absorption maximum was 0.84 at 287 nm.
between 290 nm and 360 nm. Therefore, OXB is one of the most common UV absorbers used in the sunscreen. The absorption spectrum of OXB shows the three maxima’s of absorption band shifts at: 1st ! 241 nm; 2nd ! 288 nm and 3rd ! 324 nm. The 1st and 2nd absorption bands are believed to be due to p ! p* because of the two benzene rings presents in molecular structure of OXB, whereas, the 3rd band is presumably due to the n ! p* because of the carboxyl group present in OXB molecule [6]. The sample of OXB and Met (Methionine) was continuously exposed to radiation for 3h and the exposed and unexposed samples were injected in HPLC for characterization. Fig. 3 reveals that before irradiation the chromatogram exhibited two peaks; the peak at retention time 0.8 min generated from Met, whereas, the second peak at retention time 1.25 min was produced from OXB. As
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evident from Fig. 3, no noticeable difference appeared between exposed and unexposed spectra. Is was evident that OXB proved to be highly photo-stable even in the presence of Met. Careful analysis of the HPLC chromatogram reveals a small peak at retention time 1.65 min and very small speak after the OXB peak not too much visible in the chromatogram. OXB is based on ultrafast excited state intermolecular proton transfer reaction (ESIPT) [7] and does not efficiently yield any stable photoproducts; this makes the OXB significantly photo-stable even after a prolong time of irradiation by UV-light. In ESIPT based reaction, enol form of OXB absorbs energy and then transforms into an excited state, before reverting back to the ground state by releasing its extra energy as keto emission [8]; the mechanism in shown in Fig. 4. The Figure reveals that the molecule absorbs energy to transform into singlet excited state from its ground state followed by transfer of its energy into an excitedstate molecule. The excited state is immediately quenched to ground state by internal conversion transferring back the energy of the molecule back to the OXB at ground state. OXB proved to be highly photo-stable against steady-state irradiations even at prolonged exposure for 24h at high photon flux of 80 mW, in the presence of Met; it was decided to test the system for possible transient product formation. For this investigation, nLFP was used with an excitation at 355 nm. When a high laser pulse energy was used (ca. 30–40 mJ/pulse), a rather weak transient absorption was observed being maximum at 390 nm (Fig. 5). The OXB molecule was excited by a pump pulse of 355 nm and the kinetic of the excited molecule was measured at 380 nm of probe laser as shown in Fig. 5; the figure shows kinetic trace of the OXB. The kinetic trace explains that after the excitation transient absorption decays to its initial value in a microsecond. There was no difference between the kinetic traces, in the argon saturated and oxygen saturated samples for OXB In order to observe some transient in the nLFP experiment, methionine was added as a potential scavenger of OXB-derived
Fig. 3. HPLC chromatogram of a mixture of OXB in the presence of Met (OXB 1mM + Met 10 nM) before [(black line monitored at 190 nm) and (blue line monitored at 289 nm),] and after [(red line monitored at 190 nm) and (maroon line monitored at 289 nm)]. In set the chromatogram is 2D for more clear identification of compounds present in the solution. The flow rate was 0.800 mL/min and injection volume was 20 mL. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Keto-enol tautomerization of OXB (R1 = OCH3, R2 = H) (a) and simplified ESIPT reaction mechanism (b).
transients (Fig. 5). However, after measuring the nLFP of sample in the presence of scavenger at the same experimental parameters and after normalization of the traces, no scavenging effect of the Met was observed on OXB; the decay remained approximately unchanged. That verified that OXB was highly photo-stable even in the presence of Met as quencher compound with a very less photoliability. 3.2. Avobenzone (AvoB) The UV/Vis Absorption spectroscopy of Avobenzone shows two major peaks (Fig. 6); the absorption band exhibiting maximum absorption at around 350 nm can be assigned to the enol form of AVOB while the weaker band appearing at 270 nm can be attributed to diketo form of AVOB [9]. It is evident from Fig. 6 that before exposure of the AVOB to UV light the enol form appeared to be dominant compared with diketo. Fig. 6 further reveals that the absorption band of enol decreased and that of diketo increased with exposure time. The constitutional isomer enol and diketo readily interconvert upon exposure to the photons by a chemical reaction called ‘tautomerization’. Hence, the enol and diketo can be termed as tautomer of each other in the case of AVOB. This enol to diketo migration within the molecule can be attributed to proton transfer to the adjacent double bond [10]. Various isomers show various absorption bands as the transitions of bonding orbitals to anti-bonding orbitals vary accordingly. For example, p ! p* transitions are preferable over
Fig. 5. nLFP of Oxybenzone (OXB) at 355 nm excitation wavelength without quencher (black, argon saturated solution) and with quencher added (red, 10 mM of Met) after normalization. Here, Methionine (Met) acts as a quencher. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
s ! s* since the former transitions requires lesser energy photons compared to the later. UV/Vis Absorption spectra of oxygen-saturated and argonsaturated AVOB when irradiated up to 2h, appear to be similar as shown in Fig. 6. However, prolonged exposure to UV light up to 3h generated a significant difference in the absorption bands of the AVOB both under oxygen and argon saturated conditions compared with unexposed condition (Fig. 7). That clear indication that the photoreactions of both cases were different for oxygen and argon saturated samples. Knowing the photo-unstability of AVOB, HPLC apparatus was employed in order to investigate the photoproducts generated as a result of UV irradiation. Fig. 8 reveals HPLC chromatograms of AVOB samples before and after 3h irradiation; comparison between immediately irradiated and after one day of irradiation is also demonstrated. It is evident that before irradiation only two peaks existed in the chromatogram; the higher intensity peak at retention time 1.5 min is believed to be from enol and the lower intensity peak at retention time 0.75 min is attributed to diketo. However, soon after irradiation, the enol peak intensity is decreased, whereas, intensity of diketo peak is increased. Furthermore, new peaks were observed in early retention times between 0.20 min to 0.60 min; these peaks may be evolved due to expected photo-products of AVOB. The 3h irradiated sample stored for one day revealed a changed chromatogram. The enol and diketo peaks appeared to be reversed and photo-products became stale; the enol, however, was not fully reversible as the increase was not exactly equal to the peak intensity before irradiation. This decrease in the intensity of the enol peak was expected because of stable photo-product observed in the early retention time of chromatogram. Fig. 9 displays the HPLC chromatograms of the argonsaturated samples of AVOB before and after irradiation of UVlight for 3h. Argon-saturated sample of AVOB followed similar trend as shown in Fig. 8. The enol peak decreased at retention time 1.5 min whereas the diketo peak increased at retention time 0.75 min after irradiation; few photoproducts were also observed at early retention times between 0.2–0.70 min in the chromatogram. Comparing Figs. 8 and 9, it is indicated that the photoproducts in case of argon-saturated were more compared with oxygen-saturated samples. Hence, it may be stated that degradation of AVOB in the presence of argon was more as compared to oxygen generating additional photo-products. Furthermore, in case of argon-saturated AVOB irradiated samples, the reconversion of the diketo into enol form of AVOB was not significant compared with oxygen-saturated samples; the photoproducts also appeared to be stable. Both argon-saturated and oxygensaturated solutions showed a distinct photodegradation, however, the products detected in HPLC chromatograms were noticeably different.
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Fig. 6. Absorption Specta of Avobenzone (AVOB) at various irradiation exposure (a) Oxygen-saturated (left) and (b) argon-saturated.
Fig. 7. Comparison of Absorption Spectrum of Avobenzone (AVOB) for Argon Saturated and Oxygen Saturated. Before and after irradiation at 355 nm of wavelength.
The photoproducts indicated at retention times 0.20–0.70 min in Figs. 8 and 9 for oxygen-saturated and argon-saturated samples, were collected with help of fraction collector in HPLC. The collected fractions were injected by syringe injector to LC–MS Mass Spectrometer for analytical analyses. The fraction from oxygen-saturated AVOB sample was revealed at 265.18 Da in m/z spectrum for the oxygen saturated samples the 1 fraction was collected and after injecting that fraction of LC–MS; it was the only photoproduct observed after irradiation in this case, as displayed in Fig. 10 (a). Fig. 10 (b,c) reveal m/z spectra of argon-saturated sample after irradiation; the number of peaks were more so two fractions were analysed to identify the photoproducts. One fraction displayed a dominant peak at retention time from 0.20 min to 0.50 min, at 265.18 Da similar to the oxygen-saturated sample, whereas, the second displayed a peak at 301.144 Da at retention time from 0.50 min to 0.80 min. These analyses indicated presence of two photoproducts in argon-saturated sample with molecular masses of 264.17 Da and 300.13 Da. The common photoproduct with molecular mass of 265.18 Da appearing in both oxygen- and argon-saturated samples, was
further analysed as shown in Fig. 11, however, the molecular structure could not be revealed. After establishing the photo-liability of AVOB (irradiated for 3h at high photon flux of 80 mW) by steady-state irradiation experimentation, further testing was performed on the system for possible transient product formation or triplet formation. Nanosecond laser flash photolysis (nLFP) of oxygen- and argonsaturated AVOB samples was determined at excitation wavelengths of 355 and 266 nm, as shown in Fig. 12. At excitation wavelength of 355 nm (excitation of enol), no transient absorption was observed, however, there was a bleaching (negative absorption) observed possibly due to depopulation of a ground state of AVOB. The negative signal was recovered within nanoseconds suggesting no triplet formation. The negative absorption also shows that the extinction coefficient at ground state was higher than the extinction coefficient of excited state so according to the Lambert-Beer law [11] the change in optical density should be negative. In both cases oxygen- and argon-saturated samples, the offset between the baseline and decay curve appeared because of formation of the photoproducts during excitation. Interestingly, in kinetics at excitation 355 nm (Fig. 12(a)), the argon-saturated sample showed a relatively fast decay in the beginning before coming down to a steady state approaching to the decay value of oxygen-saturated sample. This fast decay may be attributed to absorption of diketo form of AVOB. For further confirmation the system was tested for possible transient product formation or triplet formation at excitation wavelength 266 nm (Fig. 12(b)). The diketo form when excited at 266 nm, generated an intensive transient absorption signal as detected by nLFP and the lifetime of this transient was found to be dependent on the presence of oxygen. Therefore, that absorption was believed to be due to the triplet state of diketo AVOB formation. The Fig. 12(b) also shows an ultrafast triplet formation in diketo in argon-saturated sample at excitation wavelength 266 nm. Hence, it can be deduced from this experiment that AVOB usage for the sunscreens at lower wavelength are not safe. The photo-isomerization of compound was oxygen dependent occur through the triplet excited state and can be one of the rational explanation for limited stabilization of Avobenzone [12,13]. Fig. 13(a) shows transient spectrum of AVOB at 355 nm excitation wavelength; the spectrum shows that maximum light was absorbed 360 nm. The species absorbing at the wavelength of 320 nm were not significantly absorbed at the maximum depletion wavelength this depletion wavelength helped to estimate the
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Fig. 8. HPLC chromatogram of Oxygen saturated Avobenzone sample just after irradiation (a) and after one day same sample without irradiation (b). The chromatogram at 190 nm detector wavelength. The flow rate was 0.800 mL/min and injection volume is 20 mL for the analysis with mobile phase of 3:2 AcN:H2O.
Fig. 9. HPLC Chromatogram of Argon saturated Avobenzone sample just after irradiation (a) and after one day same sample without irradiation (b). The detector wavelength is 190 nm. The flow rate was 0.800 mL/min and injection volume was 20 mL for the analysis with mobile phase of 3:2 AcN:H2O.
extinction coefficient of enol rotamer form of AVOB. The extinction coefficient for rotamer was estimated about 19000 M1 cm1 10% with the help beer lambert law. As shown in Fig. 13(b) the transient spectrum of AVOB at 266 nm excitation wavelength, the maximum light was absorbed at 390 nm. It also reveals that diketo continues to absorb even at substantially longer probe wavelengths indicating a long-lived transient formation. The transient spectra from 440 to 550 nm also determines variation in optical density without approaching absolute zero. The triplet–triplet energy transfer mechanism was one of the origin of efficient triplet state population confirming the triplet state transient at 390 nm. Fig. 14 shows that singlet oxygen formation of AVOB at the excitation wavelength of 310 nm. The triplet formation was confirmed by an experiment performed in the presence of oxygen, where the singlet oxygen was observed at 1270 nm (Fig. 14 shows formation of the singlet oxygen from AVOB). The efficiency of this near-infra-red emission was found to be dependent on the oxygen
concentration as it disappeared for argon-saturated solutions. This leads to an important conclusion that formation of singlet Oxygen (through triplet–triplet energy transfer between 3AvoB* and 3O2) being a highly reactive species, may induce irreversible modifications. Singlet oxygen is an excited state of oxygen and highly energetic form of oxygen. Furthermore singlet oxygen has been found to be highly unstable and reactive radical towards organic compounds [14]. Singlet oxygen is commonly involved in damage to skin or tissues during the sunlight exposure. This can also be stated that the usage of AVOB as sunscreen above 320 nm of wavelength was safer. Further investigation on photo-reactivity of AVOB was performed using a femtosecond time resolved spectroscopy. Varying kinetics for different probe wavelengths at excitation wavelength of 355 nm are shown in Fig. 15. It is evident from the kinetics that life time for the excited species to ground state was about 40–50 ps. The remaining offset of absorption can be attributed to monophotonic ionization and deprotonation of the
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Fig. 10. LC–MS m/z Spectra for Oxygen Saturated (a) and Argon saturated samples (b) fraction for Oxygen-saturated and (c) fraction for argon-saturated samples.
AVOB molecule. This indicates that diketo form of AVOB remained excited for some time before going back to the enol stable form to the ground state. The offset in decaying can also be attributed to the photoproducts formed during excitation since all excited
Fig. 11. m/z spectra of fragmentation of 265.18 photoproduct present in both oxygen and argon saturated samples.
molecules cannot came back to their ground state and convert into stable photoproducts. 3.3. Ecamsule (ECAM) The UV/Vis absorption spectra of ECAM were determined before and after the irradiation at 355 nm using water and AcN as solvent in 2:3 ratio (Fig. 16). The ECAM before irradiation shows absorption band maximum at 350 nm with the molar absorption coefficient of 4.5 104 dm3mol1 cm1. The useful range for absorption of UV light is 300–400 nm which covers the UVA region for potential use for sunscreens. The UV/vis absorption spectra were measured after continuous irradiation for 2h at 355 nm of monochromatic UV light. As evident from Fig. 16, a significant decrease in the absorption band appeared due to photo degradation with an increase in time for irradiation, however, after 2h of irradiation no significant decrease was observed. It was also observed in Fig. 16 that the maximum of the absorption was also varying by increasing the time of irradiation. The maximum absorption appeared towards lower wavelength of 300 nm. This change in the maximum absorption also indicates formation of intermediates during degradation of the ECAM by photon excitation. It has been demonstrated [11] that the compounds which show photo induced isomerization when irradiated by UV light exhibit
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Fig. 12. Kinetics of Avobenzone (AVOB) at 355 nm (a) and at 266 nm (b) with argon and oxygen saturated. The number of averages taken during kinetic traces were 15. The solvent used AcN.
Fig. 13. Transient Absorption Spectrum of AVOB at excitation wavelength 355 nm (a) and at 266 nm (b) with different lifetime.
Fig. 14. Singlet Oxygen Formation excitation wavelength at 310 nm excitation wavelength; the spectrum was obtained from averages of 6 spectra.
interchange of isomers to one another when both the isomers have different levels of maximum absorption causing a displacement in the maximum absorption spectrum with an increase in the irradiation time. As discussed the ECAM works on the principle of reversible photo-isomerization, followed by photo excitation. ECAM absorbs UV light and releases energy in the form of heat without penetration into the skin. As the ECAM absorbs the photons of UV light molecule change from trans-isomer in to cis-isomer. The change from trans ! cis isomers being an ultrafast process, was determined using a femtosecond laser flash photolysis. Fig. 17 shows the kinetics traces of the ECAM molecule at excitation wavelength of 355 nm. It was observed that the kinetics yielded a quite short excited state (absorption maximum at 560 nm), presumably due to the initially excited singlet state with a life time of about 10 ps. The decay was substantially fast indicating that the molecules were going to their singlet excited state and coming back to ground state quickly in few picoseconds by internal conversion. In the presence
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Fig. 17. Kinetics of Ecamsule (ECAM) at Excitation wavelength of 355 nm. Kinetic trace probe wavelength 608 nm. ECAM was dissolved in AcN. Fig. 15. Femtosecond Kinetics of AVOB at various pulse wavelengths using an excitation wavelength of 355 nm. The kinetic traces were in three different probe wavelengths 450, 416 and 439 nm.
Fig. 16. UV/Vis Absorption Spectra of Ecamsule (ECAM) before and after irradiation. Before irradiation Absorbance was 0.47 at 355 nm with a maximum value of 0.61 at 340 nm. The irradiation was at 355 nm. The solvent was 3:2 AcN and H2O.
of AcN solvent ECAM showed a weak fluorescence [15] which also indicated that the molecule was going from ground state to first singlet excited state and then coming back to the ground state in few picoseconds. It decays quite fast probably due to the efficient photo-isomerization. On the other hand its photodegradation suggests that the most desired (in term of its application as a sunscreen) reversible photo-isomerization is not the only deactivation channel of its excited state. The femtosecond transient absorption spectrum of ECAM is shown in Fig. 18. The transient absorption of ECAM molecule displays absorbance of the probe pulse in the range of 500–650 nm. Interestingly displacement in the transient absorption was observed with variation in life times. The displacement in transient absorption spectrum is an evidence for vibrational cooling during depopulation of ECAM molecules at their ground states. The molecules were photoexcited with some excess vibrational energy in a solution before redistribution of energy took place at
Fig. 18. Transient Absorption Spectrum of Ecamsule (ECAM) in solvent AcN. The Excitation wavelength was 355 nm. The pulse energy was 5 mJ. The absorption was 0.5 in 2 mm of quartz cell.
vibrational manifold of the ground state. As the molecules relaxed to ground state of vibrational manifold, the energy was transferred to solvent present in excess amount releasing energy to solvent, by the process called vibrational cooling. The monitoring of the excess energy dissipation from solute to solvent play a vital role in photoinduced chemical reactions [16]. In the broader way, vibrational cooling should occur through a ladder process and by a multiphonon process. In the ladder process the vibration occur through lower order interactions with the neighbouring phonons in vibrational states of energy. The excess energy in the vibrational states fall the vibrational ladder step by step by losing the energy to phonons. In the multiphonon process, the excess energy was lost by the higher order interactions with a bunch of multiphonon states [17]. The vibrational cooling was ultrafast of the order of 10 ps. During the vibrational cooling it was quite possible that the solvent reorientation and solvent coordination (the coordination of molecule solvent H atom will change to coordination of solvent N atom) occur which was proportional to is vibrational cooling rate [18].
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4. Conclusions
Acknowlegement
Oxybenzone (OXB) proved to be a stable UV filter. Even after 24 h of irradiation there was no photodegradation of OXB even in the presence of Met as a potential quencher (except a small peak observed on a chromatogram after 24 h of irradiation). In the nLFP experiments in which Met was used as a quencher, there was no change in the kinetics of the transient decay. This suggests that although some transients are formed after OXB excitation (most likely identified as phenoxyl radicals), they are not very reactive and are not efficiently yielding any stable photoproducts. In general, the OXB sunscreen was shown to be photo-stable, however, even low concentrations of free radicals may lead to undesired adverse effects to the skin protein. AVOB appeared to be rather unstable UV sunscreen and it photodegrades with time of irradiation. However, it is a most commonly used chemical sunscreen and it is usually used in a combination with other molecules acting as AVOB photostabilizers. The post-irradiation HPLC analysis showed few prominent photoproducts on the chromatogram and also exhibited changes from enol to diketo form of AVOB. A diketo form of AVOB absorbs light at <300 nm, whereas, the enol form shows absorption at around 320–360 nm range. When the 355 nm irradiations were carried out (exciting exclusively the enol form) a prominent and irreversible photodegradation of AVOB was observed. Another reaction was a reversible phototautomarization, where the enol form was converted to the diketo form. However, a changed phenomenology was observed when the diketo form was excited at 266 nm. The intensive transient absorption signal was detected in nLFP and the lifetime of this transient was found to be dependent on the presence of oxygen. Therefore we could conclude that this absorption was due to the triplet state of diketo AVOB formation. The efficiency of the nearinfra-red emission was found to be dependent on the oxygen concentration as it disappeared completely for argon-saturated solutions. Formation of singlet oxygen may induce irreversible modifications e.g. in skin protein, leading to tissue photodegradation and other harmful effects to the skin cells. Ecamsule (ECAM) was not found to be a photo-stable sunscreen as it photodegraded with irradiation by UV light as found by the absorption spectroscopy. It decays quite fast due to the efficient photoisomerization. On the other hand, its photodegradation suggests that the most desired (in terms of its application as a sunscreen) reversible photoisomerization is not the only deactivation channel of its excited state. The other processes (triplet formation, bond breakages) remain unknown and will require further investigation. This chemical sunscreen should also be used with photo-stabilizers, otherwise it will photodegrade quickly.
I truly acknowledge the support of Erasmus Mundus Master SERP-Chem, especially Prof. Bohdan Skalski and Prof. Sandrine LACOMBE. I also thank to all my colleagues in Laboratory of Chemical Physics, Faculty of Chemistry UAM and providing me all the facilities during my research. References [1] J. Kockler, M. Oelgemöller, S. Robertson, B.D. Glass, Photostability of sunscreens, J. Photochem. Photobiol. C Photochem. Rev. 13 (2012) 91–110. [2] S. Madronich, R.L. McKenzie, L.O. Björn, M.M. Caldwell, Changes in biologically active ultraviolet radiation reaching the Earth’s surface, J. Photochem. Photobiol. B Biol. 46 (1998) 5–19. [3] M.V.R. Velasco, Broad spectrum bioactive sunscreens, Int. J. Pharm. 363 (2008) 50–57. [4] P.U. Giacomoni, L. Teta, L. Najdek, Sunscreens: the impervious path from theory to practice, Photochem. Photobiol. Sci. 9 (2010) 524–529. [5] UV Filters Chart: Sunscreen Active Ingredients | Skinacea.com. at