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Efficient laser desorption ionization mass spectrometry of polycyclic aromatic hydrocarbons using excitation energy transfer from anthracene
Chemical Physics 419 (2013) 97–100
Contents lists available at SciVerse ScienceDirect
Chemical Physics journal homepage: www.elsevier.com/locate/chemphys
Efficient laser desorption ionization mass spectrometry of polycyclic aromatic hydrocarbons using excitation energy transfer from anthracene Kensuke Fujimori, Tatsuya Fujino ⇑ Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji-shi, Tokyo 192-0397, Japan
a r t i c l e
i n f o
Article history: Available online 21 February 2013
a b s t r a c t Polycyclic aromatic hydrocarbons (PAHs), such as perylene and benzopyrene, doped at amounts on the order of femtomol (10 15 mol) in anthracene crystals could be detected by laser desorption ionization mass spectrometry. Sensitivity was roughly 103 times higher than that of LDI method in our experimental conditions. It was revealed from the excitation power dependence of the peak intensity of PAHs on the mass spectra that two-photon excitation in one UV pulse was necessary to complete the ionization process. It was also clarified that the number of defect sites that trap excitons generated in anthracene crystals could be reduced by the annealing procedure, by which an efficient energy transfer between anthracene and PAHs became possible. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are chemical compounds that have two to eight aromatic rings in their structures, and they are typically produced by natural events such as forest fires, volcanic activity, and incomplete combustion of petroleum in industrial activities. PAHs spreading in the atmosphere can seriously damage human health. For example, benzopyrene shows strong mutagenicity [1,2] and carcinogenicity [3,4]. Recently, it has been pointed out that PAHs can cause allergic diseases such as asthma and hay fever [5]. Therefore, it is important to develop useful techniques for analyzing PAHs in various environments; for this purpose, gas chromatography–mass spectrometry has been widely used because of the semivolatility of PAHs [6,7]. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is one of the powerful techniques to identify analyte molecules, as it enables the detection of analytes from their molecular weight [8–10]. MALDI consists of two important processes: ionization and desorption. Since the discovery of MALDI, several studies have been carried out to clarify the ionization process [11–15]. However, the mechanism underlying the desorption process has remained unclear. In this regard, we have recently constructed a femtosecond time-resolved mass spectrometer and proposed a possible desorption process based on exciton migration in organic crystals [16]. We used tetracene-doped anthracene (TDA) crystals as a model system of MALDI. First, anthracene molecules are photoexcited by pump pulses. Excitons generated in anthracene crystals are transferred to tetracene to produce the S1 tetracene. This excitation energy transfer was monitored by ⇑ Corresponding author. Fax: +81 42 677 2525. E-mail address: [email protected] (T. Fujino). 0301-0104/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chemphys.2013.02.014
time-resolved fluorescence spectroscopy. Then, the S1 tetracene was excited to be ion by the exciton migrated after the irradiation of probe pulses. We measured the intensity of tetracene ions as a function of delay time between the pump and probe pulses and concluded that desorption was triggered by the vibrational energy generated by the deactivation of electronically photoexcited anthracene molecules. In the above-mentioned experiments, we noted that the intensities of tetracene ions and fluorescence from the S1 tetracene were high, although the molar amount of tetracene is one-hundredth that of anthracene. Therefore, we assumed that highly sensitive detection of PAHs could be realized using PAHs molecules in anthracene crystals. In this study, we prepared perylene-doped anthracene (PDA) crystals and benzopyrene-doped anthracene (BDA) crystals for the examination of the sensitive detection of perylene and benzopyrene. These PDA and BDA crystals were subjected to laser desorption ionization (LDI) mass spectrometry using a commercial machine equipped with a nanosecond N2 laser. PAHs at amounts on the order of femtomol could be detected, and this sensitivity is roughly 103 times higher than that of LDI method in our experimental conditions. We also confirmed that the twophoton excitation condition was necessary for the ionization of PAHs in anthracene crystals. 2. Experimental procedure A benzene solution of anthracene (3.3 10 3 mol dm 3) was prepared. Benzene solutions of perylene (Per) and benzopyrene (BaP) at concentrations of 3.4 10 5, 3.4 10 6, 3.4 10 7, 3.4 10 8 and 3.4 10 9 were prepared. Aliquots of the benzene solutions of anthracene and perylene, and those of anthracene and benzopyrene were mixed in a vial, and 0.7 lL of a mixed
3. Results and discussion Fig. 1(a) shows a steady-state fluorescence spectrum of PDA crystals. The steady-state fluorescence spectra of anthracene and perylene crystals are also shown for comparison. The molar amounts of anthracene and perylene in PDA were 1.2 nmol and 12 pmol, respectively, and the molar ratio was 1:0.01. It was observed that the obtained fluorescence spectrum of PDA was not similar to those of solid anthracene and perylene. However, it resembled the fluorescence spectrum of perylene solution in benzene, as shown in Fig. 1(b), although the fluorescence peak was redshifted about 600 cm 1. Therefore, it is considered that the fluorescence of PDA is mainly from the perylene molecules almost monodispersed in anthracene crystals. Monodispersion of perylene molecules were also confirmed by PDA crystals prepared with different concentrations (supplementary data in Fig. S1). Almost the same spectra were observed, indicating monodipersion was achieved for every concentration. From Fig. 1(b), we can roughly estimate the absorbance ratio between anthracene and perylene at 337 nm by assuming the absorption spectrum of perylene in PDA is also redshifted from that in solution. Considering the molar ratio of anthracene to perylene in PDA (1:0.01), the absorbance ratio at 337 nm was estimated to be 145:1. Therefore, it can be assumed that the abundant anthracene molecules in PDA crystals mainly absorbed the photons from the excitation laser and the direct excitation of perylene was unlikely. PDA crystals were then subjected to mass spectrometry. Fig. 2(a) shows the mass spectrum of PDA crystals; the molar amounts of anthracene and perylene were 1.2 nmol and 12 pmol,
fluorescence intensity (E(λ))
(a) PDA crystals
anthracene crystals
perylene crystals
(b) PDA crystals perylene solution in benzene
400
500
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wavelength (nm) Fig. 1. Steady-state fluorescence spectrum of (a) perylene-doped anthracene (PDA) crystals. The spectra of anthracene and perylene crystals are also shown. (b) Comparison between steady-state fluorescence spectrum of PDA and that of perylene solution in benzene.
(a) PDA crystals (Ant.=1.2 nmol, Per.=12 pmol) 252 [Per]+
10 23 [Na]+ 39 [K]+
solution was dropped onto a stainless steel plate to obtain perylene-doped anthracene (PDA) or benzopyrene-doped anthracene (BDA) crystals by evaporating the solvent. Therefore, the total amounts of Per and BaP were 12 pmol, 1.2 pmol, 120 fmol, 12 fmol, and 1.2 fmol, for PDA and BDA crystals. Per crystals (Per only) or BaP crystals (BaP only) were prepared as follows: 0.35 lL of each solution with concentration of 3.4 10 5, 3.4 10 6, 3.4 10 7, 3.4 10 8 and 3.4 10 9 was dropped onto a stainless steel plate. The obtained PDA and BDA crystals were analyzed using a commercial system (MALDI micro MX, Waters, 337 nm, 2.5 ns). Each mass spectrum was obtained by averaging 200 continuous scans. Steady-state fluorescence spectra are measured with the apparatus of fluorescence microscope reported previously. PDA or BDA crystals on cover glass slip were photoexcited at 400 nm.
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excitation power (I, μJ) Fig. 2. (a) Mass spectrum of PDA crystals. (b) Excitation power dependence of peak area of [Per]+.
respectively. In the spectrum, the intense peak of the perylene cation, [Per]+, was clearly observed at m/z = 252. Although the amount of anthracene is almost 200-fold that of perylene, the small peak of the anthracene cation, [Ant]+, was observed at m/z = 178, suggesting the excitation energy transfer from anthracene to perylene, as observed in our previous study of tetracene-doped anthracene crystals. The anthracene molecules in PDA crystals absorbed photons from the excitation laser, and this excitation energy was transferred to perylene molecules by exciton migration to produce the S1 perylene. Then, the S1 perylene was excited to be ions also by the excitons generated after the initial photoexcitation. Therefore, these findings suggest that two-photon excitation in one UV pulse is necessary to complete the ionization of perylene. To confirm this argument, the excitation power dependence of the peak area of the perylene cation, [Per]+, on mass spectra is shown in Fig. 2(b). The observed peak areas are indicated by dotted circles, and the solid curve is the result of fitting analysis. The mass peak of [Per]+ was observed in the power region above 5 lJ and the peak area of [Per]+ was well reproduced by the I2 curve, where I represents the excitation laser power. Therefore, it was clarified that perylene ionization was achieved by two-photon excitation. Considering the focusing area of the excitation laser, the absorption coefficient of anthracene, and the time profile of a laser pulse, the inverse of excitation velocity was calculated by using the same method reported previously [17]; PDA crystals were photoexcited every 1.5, 1.2, 1.0. and 0.87 ns for 4, 5, 6, and 7 lJ excitation, respectively. From this calculation, two-photon excitation was found to be possible in the power region of more than 5 lJ in excitation with the laser for 2.5 ns. It is clearly observed in Fig. 2(b) that the mass peak of [Per]+ was only above 5 lJ. Next, we applied this ionization method to the sensitive detection of PAHs. Fig. 3(a) shows the laser desorption ionization (LDI) mass spectra measured with perylene crystals (perylene only) at various total amounts of perylene from 12 pmol to 1.2 fmol. Mass spectrometry was carried out with the excitation laser power of 5.8 lJ. The peak of [Per]+ was clearly observed at m/z = 252, and that of isotope was also observed at m/z = 253, when the amount
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Fig. 3. Mass spectrum of perylene crystals (perylene only) (a) at various perylene amounts from 12 pmol to 1.2 fmol. (b) Magnification of mass spectra of perylene crystals with 120, 12, and 1.2 fmol of perylene. Mass spectra of PDA crystals (c) at various perylene amounts from 12 pmol to 1.2 fmol. (d) Magnification of mass spectra of PDA crystals with 120, 12, and 1.2 fmol of perylene.
of perylene was 12 pmol. However, the peak intensity suddenly decreased when the amount of perylene was 1.2 pmol, below which, no peak of [Per]+ was observed. Fig. 3(b) shows the magnification of spectra obtained with 120, 12, and 1.2 fmol of perylene crystals, and no peak of [Per]+ was detected. One may notice that noise level in the spectrum of 12 fmol is high compared with other spectra of 120 and 1.2 fmol although experimental conditions are the same for all measurements. It might be possible to consider that vacuum level of mass spectrometer was very slightly worse than other two measurements. The detection limit of perylene can be improved by using PDA crystals. Fig. 3(c) shows mass spectra observed in the case of using PDA crystals, in which only the amount of perylene was changed whereas the amount of anthracene was fixed to 1.2 nmol for every measurement. For PDA crystals with 12 pmol of perylene, the mass peak of [Per]+ was clearly observed at m/ z = 252 and 253, although the peak intensity became almost onethird that shown in Fig. 3(a) because of the difference in absorption coefficient between anthracene and perylene. In contrast to perylene crystals in Fig. 3(a), strong peaks of perylene were observed at the amount of 1.2 pmol. The peak of perylene was observed even at a low amount. Fig. 3(d) shows the magnification of spectra for 120, 12, and 1.2 fmol of perylene, and the peak of perylene was clearly observed with a signal-to-noise ratio of more than 2. In conventional MALDI mass measurements, the detection limit of PAHs was reported to submicromolar (10 7) [18]. By using our method, however, it was clearly revealed that perylene at an amount on the order of femtomol could be observed through excitation energy transfer between anthracene to perylene. We could achieve an almost 103 -fold highly sensitive detection of perylene compared with LDI mass spectrometry performed in our experimental conditions. One may notice that the peak intensity of [Per]+ does not change proportionally to the concentration of perylene. In Fig. 1 monodispersion of perylene in PDA crystals was confirmed, however, it does not guarantee the same distance between perylene molecules in crystals. We tentatively considered that the amount of perylene within the exciton diffusion distance may not be proportional to the concentration of perylene at the low concentration limit.
The sensitive detection of PAHs using excitation energy transfer is also applicable to another compound, benzopyrene (BaP). Fig. 4(a) shows the LDI mass spectra of BaP crystals (BaP only). A strong peak of the BaP cation, [BaP]+, was observed at m/z = 252, whereas the peak of its isotope was also observed at m/z = 253 when the amount of BaP was 12 pmol. As in the case of Per, the peak intensity of [Per]+ markedly decreased when the amount of perylene became 1.2 pmol. As the magnified spectra in Fig. 4(b) shows, no peak of [Per]+ was observed at an amount below 120 fmol. By using anthracene molecules as the excitation energy donor to BaP molecules in BDA crystals, however, we realized the sensitive detection of BaP molecules. Fig. 4(c) shows the mass spectra observed from BDA crystals, in which only the amount of benzopyrene was changed whereas the amount of anthracene was fixed at 1.2 nmol for every measurement. For BDA crystals with 12 pmol of benzopyrene, the mass peak of [BaP]+ was clearly observed at m/z = 252 and 253, although peak intensity decreased compared with that shown in Fig. 3(a). When the amount of BaP was decreased, the peak intensity of [BaP]+ became small; however, the peak of [BaP]+ was clearly observed even at 1.2 fmol of benzopyrene. Therefore, the femtomol-order detection of BaP was also achieved as in the case of Per. In addition to Per and BaP, we also examined other PAH compounds, and as a result, tetracene, coronene, and benzo[ghi]perylene were detected, but not benz[a]anthracene, in the case of using anthracene as the matrix for the excitation energy donor. We consider that not only the overlap integral between the fluorescence of anthracene and the absorption of PAHs, but also the molecular structure of PAHs, which determines distance between anthracene and the analyte, determines the applicability of this method. As we mentioned above, we consider that an exciton in anthracene generated by photoexcitation is transferred to PAHs to produce the S1 PAHs, and then PAHs are ionized by another exciton generated by the initial photoexcitation. Therefore, it is assumed that efficient energy transfer between anthracene and PAHs would be hindered when the produced PDA or BDA crystals possess many defect sites. To confirm this, we carried out mass spectrometry of PDA crystals after the annealing procedure to reduce the number
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Fig. 4. Mass spectrum of benzopyrene crystals (benzopyrene only) (a) at various perylene amounts from 12 pmol to 1.2 fmol. (b) Magnification of mass spectra of benzopyrene crystals with 120, 12 and 1.2 fmol of benzopyrene. Mass spectra of BDA crystals (c) at various perylene amounts from 12 pmol to 1.2 fmol. (d) Magnification of mass spectra of BDA crystals with 120, 12 and 1.2 fmol of benzopyrene.
of defect sites. Fig. 5(a) shows the mass spectrum obtained from PDA crystals without annealing, which is basically the same as that shown in Fig. 2(a). The molar amounts of anthracene and perylene were 1.2 nmol and 12 pmol, respectively. In the mass spectrum, the peaks of [Ant]+ and [Per]+ were clearly observed. After the annealing procedure for 1 min. at 343 K, however, the peak intensity of [Ant]+ markedly decreased, whereas that of [Per]+ increased. The peak intensity ratios of [Ant]+ to [Per]+ were 1:10.6 for PDA without annealing (Fig. 5(a)) and 1:29.5 for that with annealing (Fig. 5(b)). Therefore, it was found that the number of defect sites that trap excitons generated in anthracene crystals was reduced by the annealing procedure and the efficient energy transfer between anthracene and perylene occurred. With the 5 min. annealing, as shown in Fig. 5(c), the intensities of both peaks decreased because of the evaporation of anthracene and perylene. However, the mass spectrum did not change markedly after the 5 min annealing, indi-
(a) PDA crystals (without annealing) 252 [Per]+ 35
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cating that PDA crystals with fewer defect sites were already produced during the 5 min annealing. Acknowledgements T. F. acknowledges a Grant-in-Aid for Scientific Research on Priority Area (477) from MEXT, and a Grant-in-Aid for Scientific Research (C) (No. 24550030) from JSPS. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemphys.2013. 02.014. References [1] N. Jain, V. Singla, A. Satsangi, T. Pachauri, K.M. Kumari, A. Lakhani, J. Hazard. Toxic Radioact. Waste 16 (2011) 18. [2] N. Verhofstad, C.T.M. van Oostrom, E. Zwart, L.M. Maas, J. van Benthem, F.J. van Schooten, H. van Steeg, R.W.L. Godschalk, Toxicol. Sci. 119 (2011) 218. [3] P.W. Wester, J.J.A. Muller, W. Slob, G.R. Mohn, P.M. Dortant, E.D. Kroese, Food Chem. Toxicol. 50 (2012) 927. [4] J. Wang, S. Chen, M. Tian, X. Zheng, L. Gonzales, T. Ohura, B. Mai, S.L.M. Simonich, Environ. Sci. Technol. 46 (2012) 9745. [5] K. Tohi, S. Maekawa, H. Nakase, T. Ueno, S. Tomita, J. Takeda, T. Korenaga, Y. Takahama, Proc. Jpn. Soc. Immunol. 31 (2001) 153. [6] S. Ozcan, A. Tor, M.E. Aydin, Clean Soil Air Water 37 (2009) 811. [7] M.R. Mannino, S. Orecchio, Atmos. Environ. 42 (2008) 1801. [8] K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida, Rapid Commun. Mass Spectrom. 2 (1988) 151. [9] F. Hillenkamp, M. Karas, R.C. Beavis, B.T. Chait, Anal. Chem. 63 (1991) 1193A. [10] F. Hillenkamp, M. Karas, Int. J. Mass Spectrom. 200 (2000) 71. [11] H. Ehring, M. Karas, F. Hillenkamp, Org. Mass Spectrom. 27 (1992) 427. [12] M. Karas, M. Gluckmann, J. Schafer, J. Mass Spectrom. 35 (2000) 1. [13] R. Kruger, A. Pfenninger, I. Fournier, M. Gluckmann, M. Karas, Anal. Chem. 73 (2001) 5812. [14] M. Karas, R. Kruger, Chem. Rev. 103 (2003) 427. [15] W.C. Chang, L.C.L. Huang, Y.-S. Wang, W.-P. Peng, H.C. Chang, N.Y. Hsu, W.B. Yang, C.H. Chen, Anal. Chim. Acta 582 (2007) 1. [16] Y. Minegishi, D. Morimoto, J. Matsumoto, H. Shiromaru, K. Hashimoto, T. Fujino, J. Phys. Chem. C 546 (2012) 159. [17] E.J. Heilweil, R.R. Cavanagh, J.C. Stephenson, J. Chem. Phys. 89 (1988) 5342. [18] J. Zhang, X. Dong, J. Cheng, J. Li, Y. Wang, J. Am. Soc. Mass Spectrom. 22 (2011) 1294.
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Chemical Physics journal homepage: www.elsevier.com/locate/chemphys
Efficient laser desorption ionization mass spectrometry of polycyclic aromatic hydrocarbons using excitation energy transfer from anthracene Kensuke Fujimori, Tatsuya Fujino ⇑ Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji-shi, Tokyo 192-0397, Japan
a r t i c l e
i n f o
Article history: Available online 21 February 2013
a b s t r a c t Polycyclic aromatic hydrocarbons (PAHs), such as perylene and benzopyrene, doped at amounts on the order of femtomol (10 15 mol) in anthracene crystals could be detected by laser desorption ionization mass spectrometry. Sensitivity was roughly 103 times higher than that of LDI method in our experimental conditions. It was revealed from the excitation power dependence of the peak intensity of PAHs on the mass spectra that two-photon excitation in one UV pulse was necessary to complete the ionization process. It was also clarified that the number of defect sites that trap excitons generated in anthracene crystals could be reduced by the annealing procedure, by which an efficient energy transfer between anthracene and PAHs became possible. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are chemical compounds that have two to eight aromatic rings in their structures, and they are typically produced by natural events such as forest fires, volcanic activity, and incomplete combustion of petroleum in industrial activities. PAHs spreading in the atmosphere can seriously damage human health. For example, benzopyrene shows strong mutagenicity [1,2] and carcinogenicity [3,4]. Recently, it has been pointed out that PAHs can cause allergic diseases such as asthma and hay fever [5]. Therefore, it is important to develop useful techniques for analyzing PAHs in various environments; for this purpose, gas chromatography–mass spectrometry has been widely used because of the semivolatility of PAHs [6,7]. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is one of the powerful techniques to identify analyte molecules, as it enables the detection of analytes from their molecular weight [8–10]. MALDI consists of two important processes: ionization and desorption. Since the discovery of MALDI, several studies have been carried out to clarify the ionization process [11–15]. However, the mechanism underlying the desorption process has remained unclear. In this regard, we have recently constructed a femtosecond time-resolved mass spectrometer and proposed a possible desorption process based on exciton migration in organic crystals [16]. We used tetracene-doped anthracene (TDA) crystals as a model system of MALDI. First, anthracene molecules are photoexcited by pump pulses. Excitons generated in anthracene crystals are transferred to tetracene to produce the S1 tetracene. This excitation energy transfer was monitored by ⇑ Corresponding author. Fax: +81 42 677 2525. E-mail address: [email protected] (T. Fujino). 0301-0104/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chemphys.2013.02.014
time-resolved fluorescence spectroscopy. Then, the S1 tetracene was excited to be ion by the exciton migrated after the irradiation of probe pulses. We measured the intensity of tetracene ions as a function of delay time between the pump and probe pulses and concluded that desorption was triggered by the vibrational energy generated by the deactivation of electronically photoexcited anthracene molecules. In the above-mentioned experiments, we noted that the intensities of tetracene ions and fluorescence from the S1 tetracene were high, although the molar amount of tetracene is one-hundredth that of anthracene. Therefore, we assumed that highly sensitive detection of PAHs could be realized using PAHs molecules in anthracene crystals. In this study, we prepared perylene-doped anthracene (PDA) crystals and benzopyrene-doped anthracene (BDA) crystals for the examination of the sensitive detection of perylene and benzopyrene. These PDA and BDA crystals were subjected to laser desorption ionization (LDI) mass spectrometry using a commercial machine equipped with a nanosecond N2 laser. PAHs at amounts on the order of femtomol could be detected, and this sensitivity is roughly 103 times higher than that of LDI method in our experimental conditions. We also confirmed that the twophoton excitation condition was necessary for the ionization of PAHs in anthracene crystals. 2. Experimental procedure A benzene solution of anthracene (3.3 10 3 mol dm 3) was prepared. Benzene solutions of perylene (Per) and benzopyrene (BaP) at concentrations of 3.4 10 5, 3.4 10 6, 3.4 10 7, 3.4 10 8 and 3.4 10 9 were prepared. Aliquots of the benzene solutions of anthracene and perylene, and those of anthracene and benzopyrene were mixed in a vial, and 0.7 lL of a mixed
3. Results and discussion Fig. 1(a) shows a steady-state fluorescence spectrum of PDA crystals. The steady-state fluorescence spectra of anthracene and perylene crystals are also shown for comparison. The molar amounts of anthracene and perylene in PDA were 1.2 nmol and 12 pmol, respectively, and the molar ratio was 1:0.01. It was observed that the obtained fluorescence spectrum of PDA was not similar to those of solid anthracene and perylene. However, it resembled the fluorescence spectrum of perylene solution in benzene, as shown in Fig. 1(b), although the fluorescence peak was redshifted about 600 cm 1. Therefore, it is considered that the fluorescence of PDA is mainly from the perylene molecules almost monodispersed in anthracene crystals. Monodispersion of perylene molecules were also confirmed by PDA crystals prepared with different concentrations (supplementary data in Fig. S1). Almost the same spectra were observed, indicating monodipersion was achieved for every concentration. From Fig. 1(b), we can roughly estimate the absorbance ratio between anthracene and perylene at 337 nm by assuming the absorption spectrum of perylene in PDA is also redshifted from that in solution. Considering the molar ratio of anthracene to perylene in PDA (1:0.01), the absorbance ratio at 337 nm was estimated to be 145:1. Therefore, it can be assumed that the abundant anthracene molecules in PDA crystals mainly absorbed the photons from the excitation laser and the direct excitation of perylene was unlikely. PDA crystals were then subjected to mass spectrometry. Fig. 2(a) shows the mass spectrum of PDA crystals; the molar amounts of anthracene and perylene were 1.2 nmol and 12 pmol,
fluorescence intensity (E(λ))
(a) PDA crystals
anthracene crystals
perylene crystals
(b) PDA crystals perylene solution in benzene
400
500
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wavelength (nm) Fig. 1. Steady-state fluorescence spectrum of (a) perylene-doped anthracene (PDA) crystals. The spectra of anthracene and perylene crystals are also shown. (b) Comparison between steady-state fluorescence spectrum of PDA and that of perylene solution in benzene.
(a) PDA crystals (Ant.=1.2 nmol, Per.=12 pmol) 252 [Per]+
10 23 [Na]+ 39 [K]+
solution was dropped onto a stainless steel plate to obtain perylene-doped anthracene (PDA) or benzopyrene-doped anthracene (BDA) crystals by evaporating the solvent. Therefore, the total amounts of Per and BaP were 12 pmol, 1.2 pmol, 120 fmol, 12 fmol, and 1.2 fmol, for PDA and BDA crystals. Per crystals (Per only) or BaP crystals (BaP only) were prepared as follows: 0.35 lL of each solution with concentration of 3.4 10 5, 3.4 10 6, 3.4 10 7, 3.4 10 8 and 3.4 10 9 was dropped onto a stainless steel plate. The obtained PDA and BDA crystals were analyzed using a commercial system (MALDI micro MX, Waters, 337 nm, 2.5 ns). Each mass spectrum was obtained by averaging 200 continuous scans. Steady-state fluorescence spectra are measured with the apparatus of fluorescence microscope reported previously. PDA or BDA crystals on cover glass slip were photoexcited at 400 nm.
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K. Fujimori, T. Fujino / Chemical Physics 419 (2013) 97–100
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8
excitation power (I, μJ) Fig. 2. (a) Mass spectrum of PDA crystals. (b) Excitation power dependence of peak area of [Per]+.
respectively. In the spectrum, the intense peak of the perylene cation, [Per]+, was clearly observed at m/z = 252. Although the amount of anthracene is almost 200-fold that of perylene, the small peak of the anthracene cation, [Ant]+, was observed at m/z = 178, suggesting the excitation energy transfer from anthracene to perylene, as observed in our previous study of tetracene-doped anthracene crystals. The anthracene molecules in PDA crystals absorbed photons from the excitation laser, and this excitation energy was transferred to perylene molecules by exciton migration to produce the S1 perylene. Then, the S1 perylene was excited to be ions also by the excitons generated after the initial photoexcitation. Therefore, these findings suggest that two-photon excitation in one UV pulse is necessary to complete the ionization of perylene. To confirm this argument, the excitation power dependence of the peak area of the perylene cation, [Per]+, on mass spectra is shown in Fig. 2(b). The observed peak areas are indicated by dotted circles, and the solid curve is the result of fitting analysis. The mass peak of [Per]+ was observed in the power region above 5 lJ and the peak area of [Per]+ was well reproduced by the I2 curve, where I represents the excitation laser power. Therefore, it was clarified that perylene ionization was achieved by two-photon excitation. Considering the focusing area of the excitation laser, the absorption coefficient of anthracene, and the time profile of a laser pulse, the inverse of excitation velocity was calculated by using the same method reported previously [17]; PDA crystals were photoexcited every 1.5, 1.2, 1.0. and 0.87 ns for 4, 5, 6, and 7 lJ excitation, respectively. From this calculation, two-photon excitation was found to be possible in the power region of more than 5 lJ in excitation with the laser for 2.5 ns. It is clearly observed in Fig. 2(b) that the mass peak of [Per]+ was only above 5 lJ. Next, we applied this ionization method to the sensitive detection of PAHs. Fig. 3(a) shows the laser desorption ionization (LDI) mass spectra measured with perylene crystals (perylene only) at various total amounts of perylene from 12 pmol to 1.2 fmol. Mass spectrometry was carried out with the excitation laser power of 5.8 lJ. The peak of [Per]+ was clearly observed at m/z = 252, and that of isotope was also observed at m/z = 253, when the amount
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Fig. 3. Mass spectrum of perylene crystals (perylene only) (a) at various perylene amounts from 12 pmol to 1.2 fmol. (b) Magnification of mass spectra of perylene crystals with 120, 12, and 1.2 fmol of perylene. Mass spectra of PDA crystals (c) at various perylene amounts from 12 pmol to 1.2 fmol. (d) Magnification of mass spectra of PDA crystals with 120, 12, and 1.2 fmol of perylene.
of perylene was 12 pmol. However, the peak intensity suddenly decreased when the amount of perylene was 1.2 pmol, below which, no peak of [Per]+ was observed. Fig. 3(b) shows the magnification of spectra obtained with 120, 12, and 1.2 fmol of perylene crystals, and no peak of [Per]+ was detected. One may notice that noise level in the spectrum of 12 fmol is high compared with other spectra of 120 and 1.2 fmol although experimental conditions are the same for all measurements. It might be possible to consider that vacuum level of mass spectrometer was very slightly worse than other two measurements. The detection limit of perylene can be improved by using PDA crystals. Fig. 3(c) shows mass spectra observed in the case of using PDA crystals, in which only the amount of perylene was changed whereas the amount of anthracene was fixed to 1.2 nmol for every measurement. For PDA crystals with 12 pmol of perylene, the mass peak of [Per]+ was clearly observed at m/ z = 252 and 253, although the peak intensity became almost onethird that shown in Fig. 3(a) because of the difference in absorption coefficient between anthracene and perylene. In contrast to perylene crystals in Fig. 3(a), strong peaks of perylene were observed at the amount of 1.2 pmol. The peak of perylene was observed even at a low amount. Fig. 3(d) shows the magnification of spectra for 120, 12, and 1.2 fmol of perylene, and the peak of perylene was clearly observed with a signal-to-noise ratio of more than 2. In conventional MALDI mass measurements, the detection limit of PAHs was reported to submicromolar (10 7) [18]. By using our method, however, it was clearly revealed that perylene at an amount on the order of femtomol could be observed through excitation energy transfer between anthracene to perylene. We could achieve an almost 103 -fold highly sensitive detection of perylene compared with LDI mass spectrometry performed in our experimental conditions. One may notice that the peak intensity of [Per]+ does not change proportionally to the concentration of perylene. In Fig. 1 monodispersion of perylene in PDA crystals was confirmed, however, it does not guarantee the same distance between perylene molecules in crystals. We tentatively considered that the amount of perylene within the exciton diffusion distance may not be proportional to the concentration of perylene at the low concentration limit.
The sensitive detection of PAHs using excitation energy transfer is also applicable to another compound, benzopyrene (BaP). Fig. 4(a) shows the LDI mass spectra of BaP crystals (BaP only). A strong peak of the BaP cation, [BaP]+, was observed at m/z = 252, whereas the peak of its isotope was also observed at m/z = 253 when the amount of BaP was 12 pmol. As in the case of Per, the peak intensity of [Per]+ markedly decreased when the amount of perylene became 1.2 pmol. As the magnified spectra in Fig. 4(b) shows, no peak of [Per]+ was observed at an amount below 120 fmol. By using anthracene molecules as the excitation energy donor to BaP molecules in BDA crystals, however, we realized the sensitive detection of BaP molecules. Fig. 4(c) shows the mass spectra observed from BDA crystals, in which only the amount of benzopyrene was changed whereas the amount of anthracene was fixed at 1.2 nmol for every measurement. For BDA crystals with 12 pmol of benzopyrene, the mass peak of [BaP]+ was clearly observed at m/z = 252 and 253, although peak intensity decreased compared with that shown in Fig. 3(a). When the amount of BaP was decreased, the peak intensity of [BaP]+ became small; however, the peak of [BaP]+ was clearly observed even at 1.2 fmol of benzopyrene. Therefore, the femtomol-order detection of BaP was also achieved as in the case of Per. In addition to Per and BaP, we also examined other PAH compounds, and as a result, tetracene, coronene, and benzo[ghi]perylene were detected, but not benz[a]anthracene, in the case of using anthracene as the matrix for the excitation energy donor. We consider that not only the overlap integral between the fluorescence of anthracene and the absorption of PAHs, but also the molecular structure of PAHs, which determines distance between anthracene and the analyte, determines the applicability of this method. As we mentioned above, we consider that an exciton in anthracene generated by photoexcitation is transferred to PAHs to produce the S1 PAHs, and then PAHs are ionized by another exciton generated by the initial photoexcitation. Therefore, it is assumed that efficient energy transfer between anthracene and PAHs would be hindered when the produced PDA or BDA crystals possess many defect sites. To confirm this, we carried out mass spectrometry of PDA crystals after the annealing procedure to reduce the number
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Fig. 4. Mass spectrum of benzopyrene crystals (benzopyrene only) (a) at various perylene amounts from 12 pmol to 1.2 fmol. (b) Magnification of mass spectra of benzopyrene crystals with 120, 12 and 1.2 fmol of benzopyrene. Mass spectra of BDA crystals (c) at various perylene amounts from 12 pmol to 1.2 fmol. (d) Magnification of mass spectra of BDA crystals with 120, 12 and 1.2 fmol of benzopyrene.
of defect sites. Fig. 5(a) shows the mass spectrum obtained from PDA crystals without annealing, which is basically the same as that shown in Fig. 2(a). The molar amounts of anthracene and perylene were 1.2 nmol and 12 pmol, respectively. In the mass spectrum, the peaks of [Ant]+ and [Per]+ were clearly observed. After the annealing procedure for 1 min. at 343 K, however, the peak intensity of [Ant]+ markedly decreased, whereas that of [Per]+ increased. The peak intensity ratios of [Ant]+ to [Per]+ were 1:10.6 for PDA without annealing (Fig. 5(a)) and 1:29.5 for that with annealing (Fig. 5(b)). Therefore, it was found that the number of defect sites that trap excitons generated in anthracene crystals was reduced by the annealing procedure and the efficient energy transfer between anthracene and perylene occurred. With the 5 min. annealing, as shown in Fig. 5(c), the intensities of both peaks decreased because of the evaporation of anthracene and perylene. However, the mass spectrum did not change markedly after the 5 min annealing, indi-
(a) PDA crystals (without annealing) 252 [Per]+ 35
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cating that PDA crystals with fewer defect sites were already produced during the 5 min annealing. Acknowledgements T. F. acknowledges a Grant-in-Aid for Scientific Research on Priority Area (477) from MEXT, and a Grant-in-Aid for Scientific Research (C) (No. 24550030) from JSPS. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemphys.2013. 02.014. References [1] N. Jain, V. Singla, A. Satsangi, T. Pachauri, K.M. Kumari, A. Lakhani, J. Hazard. Toxic Radioact. Waste 16 (2011) 18. [2] N. Verhofstad, C.T.M. van Oostrom, E. Zwart, L.M. Maas, J. van Benthem, F.J. van Schooten, H. van Steeg, R.W.L. Godschalk, Toxicol. Sci. 119 (2011) 218. [3] P.W. Wester, J.J.A. Muller, W. Slob, G.R. Mohn, P.M. Dortant, E.D. Kroese, Food Chem. Toxicol. 50 (2012) 927. [4] J. Wang, S. Chen, M. Tian, X. Zheng, L. Gonzales, T. Ohura, B. Mai, S.L.M. Simonich, Environ. Sci. Technol. 46 (2012) 9745. [5] K. Tohi, S. Maekawa, H. Nakase, T. Ueno, S. Tomita, J. Takeda, T. Korenaga, Y. Takahama, Proc. Jpn. Soc. Immunol. 31 (2001) 153. [6] S. Ozcan, A. Tor, M.E. Aydin, Clean Soil Air Water 37 (2009) 811. [7] M.R. Mannino, S. Orecchio, Atmos. Environ. 42 (2008) 1801. [8] K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida, Rapid Commun. Mass Spectrom. 2 (1988) 151. [9] F. Hillenkamp, M. Karas, R.C. Beavis, B.T. Chait, Anal. Chem. 63 (1991) 1193A. [10] F. Hillenkamp, M. Karas, Int. J. Mass Spectrom. 200 (2000) 71. [11] H. Ehring, M. Karas, F. Hillenkamp, Org. Mass Spectrom. 27 (1992) 427. [12] M. Karas, M. Gluckmann, J. Schafer, J. Mass Spectrom. 35 (2000) 1. [13] R. Kruger, A. Pfenninger, I. Fournier, M. Gluckmann, M. Karas, Anal. Chem. 73 (2001) 5812. [14] M. Karas, R. Kruger, Chem. Rev. 103 (2003) 427. [15] W.C. Chang, L.C.L. Huang, Y.-S. Wang, W.-P. Peng, H.C. Chang, N.Y. Hsu, W.B. Yang, C.H. Chen, Anal. Chim. Acta 582 (2007) 1. [16] Y. Minegishi, D. Morimoto, J. Matsumoto, H. Shiromaru, K. Hashimoto, T. Fujino, J. Phys. Chem. C 546 (2012) 159. [17] E.J. Heilweil, R.R. Cavanagh, J.C. Stephenson, J. Chem. Phys. 89 (1988) 5342. [18] J. Zhang, X. Dong, J. Cheng, J. Li, Y. Wang, J. Am. Soc. Mass Spectrom. 22 (2011) 1294.