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Site selection spectroscopy and photochemical hole-burning of hypericin
6 January 1995
CHEMICAL PHYSICS LETTERS
ELSEVIER
Chemical PhysicsLetters 232 (1995) 127-134
Site selection spectroscopy and photochemical hole-burning of hypericin S . M . A r a b e i a,l, j . p . G a l a u p a,., p. J a r d o n b "Laboratoire de Photophysique et de Photochirnie des Matbriaux Moldculaires, Ecole Normale Supbrieure de Cachan, 61, Avenue du Prdsident Wilson, 94235 Cachan Cedex, France b Laboratoire de Photochimie et Electrochirnie Molbculaire, Universitb Joseph Fourier, BP 68, 38402 St. Martin d'Hbres Cedex, France
Received 11 July 1994; in final form 3 November 1994
Abstract
High-resolution fluorescence and excitation spectra of hypericin in ethanol and in polyvinylbutiral are reported. Persistent spectral hole-burning is highly efficient in ethanol with a quantum yield q~hb~ 1.1 × 10- 2. Evidence is shown for a photoproduct located at about 200 cm- ~below the burnt hole. Possible photochemical reactions are discussed.
1. Introduction
The molecule ofhypericin (see the insert in Fig. 1 ) is a natural pigment which can be extracted from plants or insects where it plays a role as a photoreceptor [ 1,2 ]. It has also been discovered in some fossils [ 3 ]. Its phototoxicity has been known for a long time. It is probably determined mainly by the high quantum yield of hypericin photosensitizing the formation ofsinglet oxygen. A quantum yield of ~ 0.73 has been measured for the production of singlet oxygen in organic solutions [4,5] (this value decreases to ~0.35 in liposomic media [6] ). It may be because of this property that hypericin has been introduced in the photodynamic therapy of cancer tumours [ 7 ], as an anti-virus agent [ 8,9 ] and it has also been proposed to help in the fight against AIDS [ 10]. The spectral properties of hypericin have been * Corresponding author. J Permanent address: Institute of Molecular and Atomic Physics, Academy of Science of Belarus', 70, F. Skarina Avenue, 220072 Minsk (Belarus').
studied in detail (absorption and luminescence) only in organic solutions at room temperature or at liquid nitrogen temperature [ 11,12 ]. The photophysical parameters of the lowest singlet and triplet states, $1 and T1 have been determined for different solvents [ 12-16 ]; also, the characteristics of the triplet-triplet absorption spectra have been established [ 12-14 ]. Nevertheless, precise data on the vibronic structure of the ground state and the lowest electronic excited state of hypericin are lacking in the literature. Measurements performed at room or at liquid nitrogen temperatures are limited because of the large homogeneous broadening at these temperatures and because of the non-elimination of the inhomogeneous broadening (no selective excitation). In these conditions, most of the vibration bands are not resolved in several fine lines. More precise data come from resonance Raman (RR) spectra which have been recorded in a limited range, 600-1700 c m - i [ 17 ]. Persistent spectral hole-burning results have been reported recently [18]: the authors studied the influence of pressure on the hole shape; among other
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S.M. Arabei et al. / Chemical Physics Letters 232 (1995) 127-134
128
H 0S
~
H~ 0
2. Experimental
/
0
CHa
NO HO
CH~
P
i 1
d
I ,^,
i t / ./
400
500
r 700
600
~,,
b
o.
,J 600
650
700
~-,
Fig. 1. Hypericin in ethanol: absorption spectrum and fluorescence spectrum under excitation with all argon laser lines at 293 K; (a) fluorescence excitation spectrum (,~nuo>645 nm) and fluorescence spectrum under excitation with all argon laser lines at 4.2 K (b).
molecules, hypericin was used as a probe in a 3:1 mixture of ethanol/methanol at 1.5 K. The aim of this Letter is to report the high-resolution fluorescence and excitation spectra of hypericin in an organic glass of ethanol at liquid helium temperature. We also report spectral hole-burning measurements. Spectral characteristic of burnt holes are established for hypericin in ethanol glass and in a polymer film of polyvinylbutiral (PVB). Evidence is found for a photochemical reaction and a photoproduct is localized. We discuss the possible photochemical changes occurring for hypericin in these hosts.
The hypericin used in this study has been synthesized and purified in the Laboratoire de Photochimie et Electrochimie Molrculaire at the University Joseph Fourier in Grenoble according to a procedure described previously [ 19 ]. Spectroscopic grade ethanol has been used as a solvent at room temperature or as a transparent glass matrix at 4.2 K. We also prepared doped films of polyvinylbutiral (PVB, from Aldrich) in the usual way: PVB and hypericin were dissolved in ethanol and a drop of the solution was poured on a glass substrate. After natural evaporation of the solvent at room temperature, a solid film was obtained. Separation from the glass substrate was achieved by immersion in liquid nitrogen. A concentration of 10 -4 mol £-~ has been used in all samples (in solution as well as in films). A free-running ring dye laser pumped with an Ar + ion laser (Spectra Physics, model 2016, 5 W all lines) was used for fluorescence excitation experiments in the $1 ,-So absorption band. Rhodamine 6G was the dye used to tune the laser frequency over the range 570-605 nm with a laser line width of the order of 1 cm-1. The fluorescence wavelength was selected using a monochromator (Jobin-Yvon, HRS) allowing a maximal spectral resolution of 0.4 A. The signal delivered by a photomultiplier (Hamamatsu R943-02 ) was amplified in a picoammeter (Keithley, model 485) and transferred into a computer. The site-selected fluorescence signals were recorded in the following way: while scanning the monochromator in order to record the fluorescence spectrum, we scanned the laser excitation frequency at low speed over a narrow range (approximately 3.5 nm). In this way, we could avoid the change induced in the absorbance ofhypericin due to spectral hole-burning when exciting the sample at a fixed wavelength. The data recorded in these conditions were corrected by software to remove the influence of the laser excitation frequency scanning. Fluorescence excitation spectra were obtained by scanning the laser excitation wavelength. In holeburning spectra, the broad band fluorescence was collected through a cut-off filter (all 2nuo> 645 nm). For site selection excitation spectra, the fluorescence was selectively recorded at a fixed wavelength behind the monochromator. Room temperature absorption
S.M. Arabei et aL / Chemical Physics Letters 232 (1995) 127-134
measurements have been performed with a conventional spectrofluorimeter (Philips, model PU 8720). The samples were placed inside a liquid helium bath cryostat with variable temperature facilities (SMC, FAir Liquide ). 3. Results and discussion
3.1. Absorption and fluorescence spectra at 300 and 4.2K Room temperature absorption and fluorescence spectra of hypericin in ethanol are shown in the Fig. la. The mirror symmetry rule for the fluorescence spectrum with respect to the absorption spectrum in the range 500-600 nm is fulfilled. This proves that the absorption observed in this spectral range is only due to the vibronic transitions of the $1 state. Nevertheless, a careful analysis shows that the vibrational frequencies measured in the excited S~ state (Avs,, in the absorption spectrum) are larger than the vibrational frequencies measured in the ground state (Avso, in the fluorescence spectrum). For instance, we measured a difference A Vs, - A Vso= 120 c m - 1for the high frequency vibronic band at 2600 cm-1 of hypericin in ethanol. This difference is also larger for the same bands in the absorption and fluorescence spectra of hypericin in the PVB film at 300 K (AVsl -Avso = 170 cm - I ). The 0-0 band of the fluorescence spectra of hypericin in ethanol is practically unshifted when the temperature is lowered to 4.2 K (see Fig. lb). However, the vibronic bands of the ground state are red-shifted by 2-4 nm, increasing the frequency intervals A Vso. A similar observation has been reported for the fluorescence of hypericin in ethanol at liquid nitrogen temperature [ 12]. The absorption spectrum (more precisely, the fluorescence excitation spectrum) is also red-shifted by 3.2 nm from room to liquid helium temperature (see Fig. lb). In this situation, we find that the vibrational frequencies are nearly equal in the ground state and in the first excited singlet state. For instance, the band at 578.2 nm in the fluorescence excitation spectrum assigned to a vibrational mode at Avs, =450 cm - l corresponds to a band at 610.5 nm in the fluorescence spectrum giving the same vibrational frequency A Vso= 450 c m - 1. At room temperature, the vibrational frequencies
129
are larger in the excited state than in the ground state. As also pointed out here, this difference disappears at low temperature. This observation perhaps reveals the occurrence of a possible dynamic equilibrium between different tautomers of hypericin (see further on) at room temperature. This equilibrium will be temperature dependent, resulting in the fact that one configuration only is expected to be populated at low temperature. A part of the high resolution site-selected fluorescence spectrum of hypericin in ethanol at 4.2 K is shown in Fig. 2a (curve ( 2 ) ) . The narrow dye laser excitation is located on the red edge of the inhomogeneous absorption band S 1~- So (during the registration of the fluorescence, the wavelength of the excitation laser was scanned from 596.7 to 593.3 nm). Despite the selective excitation with a narrow laser line, the vibronic band of the fluorescence spectrum around 703.2 nm (not shown in the figure) remains large and structureless. The average frequency interval for this band is large ( ~ 2608 c m - 1). A possible assignment for this band is to - O H stretching vibrations involved in hydrogen bonds, intramolecular or between guest and host. These vibrations are expected to be sensitive to local changes. We think that
i
'
570
'
~o
'
'
580
12bo
'
'
,,bo
590
'~,~-,
;% n m
Fig. 2. Fluorescence (a) and fluorescenceexcitation (b) spectra of hypericin in ethanol at 4.2 K. On top (a), curve ( 1): fluorescence using non-selective excitation with all argon laser lines; curve (2): fluorescenceunder selectiveexcitation with a monochromatic dye laser line (596.7-593.3 nm, Ave~c.~l cm-', Avr~ 1 cm-l).
130
S.M. Arabei et al. / Chemical Physics Letters 232 (1995) 127-134
this is a reason why, despite the selective laser excitation, the v i b r a t i o n a l f r e q u e n c i e s can be different resulting in a lack o f c o r r e l a t i o n b e t w e e n the S~ ( 0 ) So(v) intervals for centres excited t h r o u g h the channel S~ ( 0 ) ,-- So ( 0 ) . F o r this b a n d , the selective excitation is u n a b l e to e l i m i n a t e c o m p l e t e l y the i n h o m o geneous b r o a d e n i n g . I f the r e a s o n for the b r o a d e n i n g was s o m e v i b r o n i c congestion, we should observe this b a n d at low t e m p e r a t u r e at least partially resolved. We do not o b s e r v e a decrease in the f l u o r e s c e n c e intensity o f h y p e r i c i n u n d e r e x c i t a t i o n w i t h argon laser light at 4.2 K. In this case, p h o t o p h y s i c a l and p h o t o c h e m i c a l changes i n v o l v i n g h y p e r i c i n are in a stable e q u i l i b r i u m s i t u a t i o n r e a c h e d b e t w e e n direct a n d reverse l i g h t - i n d u c e d reactions. T h e fluorescence s p e c t r u m c o v e r i n g the range 5 9 5 . 0 - 6 8 5 . 0 n m for h y p e r i c i n u n d e r argon laser i r r a d i a t i o n at 4.2 K is s h o w n in Fig. 2a ( c u r v e ( 1 ) ). In this figure, we c o m pare the b r o a d b a n d fluorescence w i t h the site-selected fluorescence: as o b s e r v e d , the intensity distrib u t i o n a m o n g the b r o a d b a n d s is similar to the intensity d i s t r i b u t i o n a m o n g the fine lines o f the resolved spectrum. T h i s o b s e r v a t i o n p r o v e s that the m e t h o d used to r e c o r d the site-selected fluorescence, s c a n n i n g s i m u l t a n e o u s l y the e x c i t a t i o n f r e q u e n c y o v e r a small range, is efficient in e l i m i n a t i n g any influence o f a persistent h o l e - b u r n i n g m e c h a n i s m . T h e site-selected e x c i t a t i o n s p e c t r u m o f h y p e r i c i n in e t h a n o l s h o w n in Fig. 2b was o b t a i n e d r e c o r d i n g the fluorescence o v e r a n a r r o w spectral range a r o u n d 595 nm. T h e accessible spectral range is l i m i t e d to 0 750 c m - ' because o f the s c a n n i n g capabilities o f the dye used. All the vibrational frequencies m e a s u r e d for the g r o u n d state a n d the lowest e l e c t r o n i c state are p r e s e n t e d in T a b l e 1. F o r c o m p a r i s o n , we also s h o w the c o r r e s p o n d i n g r e s o n a n c e R a m a n d a t a p u b l i s h e d in the literature [ 17 ]. T h e m i r r o r s y m m e t r y b e t w e e n the fluorescence and the a b s o r p t i o n spectra is well p r e s e r v e d . We notice that the m o s t intense lines b e l o n g i n g to the 0 - 7 5 0 c m -~ range in the fluorescence s p e c t r u m (451 a n d 463 c m - i ) are not r e s o l v e d in the fluorescence excit a t i o n s p e c t r u m ( 4 5 0 c m - 1). S o m e d i s c r e p a n c i e s are o b s e r v e d b e t w e e n o u r results a n d the r e s o n a n c e Ram a n d a t a [ 17 ]: the difference can be as large as 13 cm-' for s o m e v i b r a t i o n lines ( f o r instance: A v s o = 1305 c m -1 a n d AVRR = 1292 cm -~, 6 = -- 13 c m - ' , o r also A v s o = 1647 c m - t a n d AVRR= 1636
Table 1 Vibrational frequencies (in cm -I ) of hypericin in ethanol in the ground state (AVso) and in the first excited singlet state (AVs,), compared to resonance Raman data (Avv.x) Avso
Avs~
143 226 298 301 351
384 401 451 463 490 520 580 608 627 649 685 705 752 903 929 948
145 220 296 305
-
318
-
340 376 400 422 436
-
1330
482 505 576 622 623 643
1471 1557 1601 1647 1705 1751 1761 1794 1829 1931 ~2608
627 -
690
693
748 -
920
-
-
1119 -
1351 1378 1402
-
450
1098
1136 1195 1257 1305
AVRR [ 17]
-
-
1245 1292 1313 1328 1370 1456 1499 1557 1596 1636 -
S.M. Arabei et al. / Chemical Physics Letters 232 (1995) 127-134
c m - ~, J = - 11 c m - ~). Also, some lines show different activities: for instance, the line at 1305 cm-1 in the site-selected fluorescence spectrum of hypericin has a large intensity compared to the corresponding line at 1292 c m - t which appears with a weak intensity in the resonance Raman spectrum. These differences are probably due to the fact that we compare two very different systems and experimental conditions: low temperature fluorescence data ofhypericin in ethanol glass on one hand, Raman data on a pure solid ofhypericin at room temperature on the other. A complete assignment together with a normal mode analysis of hypericin will be published in a forthcoming paper.
131 1.0
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3.2. Persistent spectral hole-burning
T 0 ~.,rl/tt
b Ethanol
08 0.8
Efficient persistent spectral hole-burning is observed for hypericin in ethanol glass as well as in PVB films. Some results are shown in Fig. 3 (top). In this work, we are interested in the stability of persistent holes and also in the photochemical hole-burning mechanism. We burnt deep holes in the inhomogeneous absorption band of hypericin in ethanol (Fig. 3a ( top ) ) or in the PVB film (Fig. 3 b ( t o p ) ) using ~ 1 mW cm -2 for 1000 s at 5.0 K. At the laser excitation frequency, the optical density decreased more than 70% in ethanol glass and more than 60% in the PVB film. In both matrices, a pseudo-phonon side hole is recorded with a maximum located at 18 cm-~ on the red-side from the narrow zero-phonon hole. A much weaker phonon side band is also detected on the blueside with a broad maximum at approximately 12 c m - i.
At the laser excitation frequency, a decrease of 40%-45% in the optical density at 5.0 K is performed with a burning time of 45 s with the following condilions: p~,th= 1 mW cm -2 at 2~,th =595.0 nm and P ~ W = l mW cm -2 at 2bPW=598.9 nm (Fig. 3a (middle)). Then, the burning process slows down and 65% of complete saturation is achieved after 10 min irradiation. The kinetics of hole growing at the early stage of burning is shown in Fig. 3b(middle). The hole-burning efficiency is higher in the ethanol glass than in the polymeric host. Despite the saturation of the zero-phonon hole, we notice that the growing of the pseudo-phonon side hole does not slow
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\ 0
2
4
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10
20
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o 50
40 30 2C I0
o
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a
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/ 30
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Fig. 3. (top) Hole-burning spectra of hypericin in ethanol (a) and in PVB (b) at 5.0 K (Wex~0.1 ~tW cm -~, 2nuo> 645 nm). Hole-burning conditions were: (a) 2b= 595.0 rim, tb= 1000 S, Wb~l mW cm -2, Tb~5.0 K, (b) 2b=598.9 nm, tb=1000 S, Wb~ 1 mW cm -2, Tb~ 5.0 K. (middle) Relative decrease of the absorption of hypericin in ethanol at 595.0 nm (a) and (b) and in PVB at 598.9 nm (a) at 5.0 K versus burning time ( Wb~ 1 mW cm-2). (bottom) Hole width versus temperature for hypericin in ethanol and in PVB.
down. This is understandable because this pseudoband is due to the burning of centres located on the red-side with respect to the laser excitation frequency
132
S.M. Arabei et al. /Chemical Physics Letters 232 (1995) 127-134
and which are excited through their phonon side band [20 ]. As the absorption in the phonon wing is weaker (see Fig. 3 (top)), the burning kinetic will be slower than for the zero-phonon hole. The hole-burning quantum efficiency is calculated according to the following equation [ 21 ]:
~hb=
[d( - ALl)/dt]t=o AOJh/Ao)i 103 Io( 1 _ 10-AO)~p '
where A is the absorption, Cp the molar extinction coefficient, Io the incident laser intensity on the sample, and AWh and Ao~i the homogeneous and inhomogeneous bandwidths, respectively. Using the data shown in Fig. 3b(middle), we estimate 4~hb~ 1.1×10 -2 which represents quite a high value. For comparison, the following values have been reported in the literature: for instance, for quinizarin in EtOH:MeOH glasses, q~hb~ 10 -4 [22], for some porphyrins, q~hb~ 10- 3 [ 21 ]. Higher hole-burning quantum yields have been evaluated only for twocolour photon gated hole-burning processes. As an example of an efficient two-photon gated spectral hole-burning system, we can mention the case ofperylene in boric acid glass for which a value q~hb~ 10has been estimated [23 ]. In Fig. 3 (bottom), we present a qualitative result showing the increase in the hole width with temperature for hypericin in ethanol and in PVB. For these experiments, the burning time has been changed from 1 to 5 min and the burning power from 1 to 10 mW cm-2 when the temperature was increased. The solid line results from an interpolation between the experimental points using a polynomial expression: it is presented here only for the sake of clarity of the figure. A precise fit of these data will have no simple meaning because hole widths were measured on nearly saturated holes. In these conditions, we cannot get the change in the homogeneous width with temperature. Nevertheless, as shown in the figure, the holes are broader in ethanol glass than in the polymeric host, even at 4.2 K; they also widen more rapidly. The fact that the holes are broader in ethanol than in a polymeric host has been reported several times in the literature, for different guest molecules, independently of the hole-burning mechanism, intramolecular or intermolecular photochemical hole-burning or non-photochemical hole-burning [ 24-26 ]. The
influence of hydrogen bonding between hypericin and the alcoholic host should be investigated, for instance, using deuterated ethanol as the host. This has not been done, but from the literature [25], it appears that proton tunnelling between hydrogen bonds, in the host or between guest and host, do not seem to be responsible for dephasing (and then for the hole width) as proved by the fact that no change is observed in the hole width when normal ethanol is replaced by deuterated ethanol in the case of resorufin. Conversely, deuteration has a drastic effect on the hole-burning efficiency which is strongly decreased in the deuterated host. From a different approach, differences in the hole widths between ethanol and polymeric hosts have been assigned to differences in the coupling constants between guest and host [ 27 ]. 3.3. Hypericin photoproduct
The presence of several hydroxyl groups at the periphery of the hypericin molecule suggests that a similar mechanism as the one suspected to explain the photochemical hole-burning of quinizarin in alcoholic solvents can also be assumed in this case. In the literature, two possible photo-rearrangements are discussed for quinizarin: the intramolecular proton transfer of one or two protons (tautomerization) and the occurrence of hydrogen bonding with the host (conformational changes) [ 22,28-31 ]. For these two cases, photoinduced changes in the hydrogen bonds of quinizarin involving one or two hydroxyl groups have been observed. In this work, we have made a systematic study of the changes in the excitation spectra (which is proportional to the absorption profile) of hypericin in ethanol with the increase in burning time, at a fixed frequency arbitrarily chosen at 595.0 nm in our experiment. We observed that the increase in the zerophonon hole depth and the pseudo-phonon hole depth induces the growth of a new absorption band on the red-side, around 600-603 nm (see Fig. 4). The superposition of all the fluorescence excitation spectra we have clearly shows an isobestic point at 598.5 nm. This point proves the existence of a spectral form of a photoproduct of hypericin which should result from a unique photoinduced change. When several spectral holes are burnt in the blue spectral range 594.3595.1 nm (Fig. 4, curve (4)), a corresponding blue-
133
S.M. Arabei et al. / Chemical Physics Letters 232 (1995) 127-134 i M .x
"-... \ "...\ ....\
%N o.
o
"n °-n-'°
I_ 3
Fig. 4. Fluorescence excitation spectra of hyperiein in ethanol at 5.0 K ( W ~ 0 . 1 ~tW cm -2, ,taro> 645 nm) after burning a hole at 2b=595.0 nm with Wb~l mWcm-2 and tb=0 S (1), tb=225 S ( 2 ), tb= 1600 S (3). The spectrum (4) has been recorded after burning several holes with the followingconditions: 2b= 595.0594.4 nm, tb=900 S, Wb~ 1 mW cm -2. shift is also observed in the absorption band of the photoproduct. The fluorescence excitation spectrum o f the photoproduct has two maxima: the first one is shifted to the red by 227 cm -~ from the position of the zero-phonon hole; the second one is located, not very clearly, in a 150-200 c m - 1 area, also on the redside. The existence o f a broad band corresponding to the photoproduct proves that the main photochemical reaction responsible for the hole-burning mechanism must be a tautomerization reaction. The reason is as follows. A careful analysis shows that all the possible intramolecular transfers of one or two protons of hypericin are accompanied by drastic changes in the conjugated electronic n system. In the insert of Fig. 4, we have drawn the four different conjugated electronic n systems of hypericin associated with possible tautomeric changes. In total, sixteen different configurations should be drawn, but most of them are equivalent and should correspond approximately to the same energies. These changes explain how different photoproducts absorbing at different frequencies can be produced. This situation differs from that of quinizarin because similar drastic changes in its conjugated system are not possible and a photoproduct cannot be definitely identified. As pointed out previously, these spectral forms probably exist also in dynamic equilibrium at room temperature. Nevertheless, we must add that the spectral forms ofhypericin located on the low energy side and which
appear during the photochemical hole-burning process are not unique. Our experiences show that the hole burnt at 595.0 nm is refilled partly if another hole is burnt on its blue-side (594.8 nm). This second hole also decreases when a new hole is burnt at higher energy (594.6 n m ) , and so on .... Fig. 5 describes these experiments: successive holes are labelled by numbers from 1 to 7 according to their chronological order. A spectral analysis of the complete hole shape shows that there are also other forms of photoproducts which are located on both sides of the burnt hole (the average spectral distribution for such photoproducts is estimated to be extended over -k-_7 cm-~ around the burning wavelength. This feature is characteristic of non-photochemical holeburning mechanisms thought to be only due to some
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---
V
J
--.
.
ii
ii~t
413,211
,7',6',s i i 594
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Fig. 5. Successive hole-burning in the absorption hand of hyper-
icin in ethanol at 5.0 K (tb=900 s, Wb~ I mW cm-2).
134
S.M. Arabei et al. /Chemical Physics Letters 232 (1995) 12 7-134
rearrangements in the host cage around the guest molecule. Nevertheless, this is not a definite proof because hypericin has the possibility of forming intermolecular hydrogen bonds with the host. Possible laser-induced changes among these hydrogen bonds would be a photochemical mechanism which would not involve a modification of the electronic conjugated system and then no drastic energy shifts.
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[ 13] P. Jardon and R. Gautron, J. Chim. Phys. 86 (1989) 2173. [14] A. Michaeli, A. Regev, Y. Mazur, J. Feitelson and H. Levanon, J. Phys. Chem. 97 (1993) 9154. [ 15 ] T. Yamazaki, N. Ohta, I. Yamazaki and P.-S. Song, J. Phys. Chem. 97 (1993) 7870. [16] E.B. Walker, T.Y. Lee and P.-S. Song, Biochim. Biophys. Acta 587 (1979) 129. [17]L.N. Raser, S.V. Kolaczkowski and T.M. Cotton, Photochem. Photobiol. 56 (1992) 157. [ 18 ] H. Pschierer, J. Friedrich, H. Falk and W. Schmitzberger, J. Phys. Chem. 97 (1993) 6902. [ 19 ] H. Brockman, K. Kluge and M. Muxfeldt, Chem. Ber. 90 (1957) 2302. [20] I.J. Lee, J.M. Hayes and G.J. Small, J. Chem. Phys. 91 (1989) 3463. [21 ] K. Horie, M. Ikemoto, T. Suzuki, S. Machida, T. Yamashita and N. Murase, Chem. Phys. Letters 195 (1992) 563. [22] Y. Iino, T. Tani, M. Sakuda, H. Nakahara and K. Fukuda, Chem. Phys. Letters 140 (1987) 76. [23]E.I. Alshits, B.M. Kharlamov and R.I. Personov, Opt. Spectry. 65 (1989) 326. [ 24 ] H.P.H. Thijssen, A.I.M. Dicker and S. V~51ker,Chem. Phys. Letters 92 (1982) 7; H.P.H. Thijssen, R. Van der Berg and S. V61ker, Chem. Phys. Letters 97 (1983) 295. [25] M. Berg, C.A. Walsh, L.R. Narasimhan, K.A. Littau and M.D. Fayer, J. Chem. Phys. 88 (1988) 1564. [26] W. Breinl, J. Freidrich and D. Haarer, Phys. Rev. B 34 (1986) 7271. [27] H.P.H. Thijssen, R. Van den Berg and S. V61ker, Chem. Phys. Letters 120 (1985) 503. [28] F. Graf, H.K. Hong, A. Nazzal and D. Haarer, Chem. Phys. Letters 59 (1978) 217. [29] J. Friedrich and D. Haarer, J. Chem. Phys. 76 (1982) 61. [30] T. Tani, Y. Sahakibara, Japan J. Appl. Phys. 31 part 1 (1992) 703. [31 ] T. Tani and A. Itami, Mol. Cryst. Liquid Cryst. 216 (1992) 247.
CHEMICAL PHYSICS LETTERS
ELSEVIER
Chemical PhysicsLetters 232 (1995) 127-134
Site selection spectroscopy and photochemical hole-burning of hypericin S . M . A r a b e i a,l, j . p . G a l a u p a,., p. J a r d o n b "Laboratoire de Photophysique et de Photochirnie des Matbriaux Moldculaires, Ecole Normale Supbrieure de Cachan, 61, Avenue du Prdsident Wilson, 94235 Cachan Cedex, France b Laboratoire de Photochimie et Electrochirnie Molbculaire, Universitb Joseph Fourier, BP 68, 38402 St. Martin d'Hbres Cedex, France
Received 11 July 1994; in final form 3 November 1994
Abstract
High-resolution fluorescence and excitation spectra of hypericin in ethanol and in polyvinylbutiral are reported. Persistent spectral hole-burning is highly efficient in ethanol with a quantum yield q~hb~ 1.1 × 10- 2. Evidence is shown for a photoproduct located at about 200 cm- ~below the burnt hole. Possible photochemical reactions are discussed.
1. Introduction
The molecule ofhypericin (see the insert in Fig. 1 ) is a natural pigment which can be extracted from plants or insects where it plays a role as a photoreceptor [ 1,2 ]. It has also been discovered in some fossils [ 3 ]. Its phototoxicity has been known for a long time. It is probably determined mainly by the high quantum yield of hypericin photosensitizing the formation ofsinglet oxygen. A quantum yield of ~ 0.73 has been measured for the production of singlet oxygen in organic solutions [4,5] (this value decreases to ~0.35 in liposomic media [6] ). It may be because of this property that hypericin has been introduced in the photodynamic therapy of cancer tumours [ 7 ], as an anti-virus agent [ 8,9 ] and it has also been proposed to help in the fight against AIDS [ 10]. The spectral properties of hypericin have been * Corresponding author. J Permanent address: Institute of Molecular and Atomic Physics, Academy of Science of Belarus', 70, F. Skarina Avenue, 220072 Minsk (Belarus').
studied in detail (absorption and luminescence) only in organic solutions at room temperature or at liquid nitrogen temperature [ 11,12 ]. The photophysical parameters of the lowest singlet and triplet states, $1 and T1 have been determined for different solvents [ 12-16 ]; also, the characteristics of the triplet-triplet absorption spectra have been established [ 12-14 ]. Nevertheless, precise data on the vibronic structure of the ground state and the lowest electronic excited state of hypericin are lacking in the literature. Measurements performed at room or at liquid nitrogen temperatures are limited because of the large homogeneous broadening at these temperatures and because of the non-elimination of the inhomogeneous broadening (no selective excitation). In these conditions, most of the vibration bands are not resolved in several fine lines. More precise data come from resonance Raman (RR) spectra which have been recorded in a limited range, 600-1700 c m - i [ 17 ]. Persistent spectral hole-burning results have been reported recently [18]: the authors studied the influence of pressure on the hole shape; among other
0009-2614/95/$09.50 © 1995 ElsevierScienceB.V. All fights reserved SSDI0009-2614(94)01324-1
S.M. Arabei et al. / Chemical Physics Letters 232 (1995) 127-134
128
H 0S
~
H~ 0
2. Experimental
/
0
CHa
NO HO
CH~
P
i 1
d
I ,^,
i t / ./
400
500
r 700
600
~,,
b
o.
,J 600
650
700
~-,
Fig. 1. Hypericin in ethanol: absorption spectrum and fluorescence spectrum under excitation with all argon laser lines at 293 K; (a) fluorescence excitation spectrum (,~nuo>645 nm) and fluorescence spectrum under excitation with all argon laser lines at 4.2 K (b).
molecules, hypericin was used as a probe in a 3:1 mixture of ethanol/methanol at 1.5 K. The aim of this Letter is to report the high-resolution fluorescence and excitation spectra of hypericin in an organic glass of ethanol at liquid helium temperature. We also report spectral hole-burning measurements. Spectral characteristic of burnt holes are established for hypericin in ethanol glass and in a polymer film of polyvinylbutiral (PVB). Evidence is found for a photochemical reaction and a photoproduct is localized. We discuss the possible photochemical changes occurring for hypericin in these hosts.
The hypericin used in this study has been synthesized and purified in the Laboratoire de Photochimie et Electrochimie Molrculaire at the University Joseph Fourier in Grenoble according to a procedure described previously [ 19 ]. Spectroscopic grade ethanol has been used as a solvent at room temperature or as a transparent glass matrix at 4.2 K. We also prepared doped films of polyvinylbutiral (PVB, from Aldrich) in the usual way: PVB and hypericin were dissolved in ethanol and a drop of the solution was poured on a glass substrate. After natural evaporation of the solvent at room temperature, a solid film was obtained. Separation from the glass substrate was achieved by immersion in liquid nitrogen. A concentration of 10 -4 mol £-~ has been used in all samples (in solution as well as in films). A free-running ring dye laser pumped with an Ar + ion laser (Spectra Physics, model 2016, 5 W all lines) was used for fluorescence excitation experiments in the $1 ,-So absorption band. Rhodamine 6G was the dye used to tune the laser frequency over the range 570-605 nm with a laser line width of the order of 1 cm-1. The fluorescence wavelength was selected using a monochromator (Jobin-Yvon, HRS) allowing a maximal spectral resolution of 0.4 A. The signal delivered by a photomultiplier (Hamamatsu R943-02 ) was amplified in a picoammeter (Keithley, model 485) and transferred into a computer. The site-selected fluorescence signals were recorded in the following way: while scanning the monochromator in order to record the fluorescence spectrum, we scanned the laser excitation frequency at low speed over a narrow range (approximately 3.5 nm). In this way, we could avoid the change induced in the absorbance ofhypericin due to spectral hole-burning when exciting the sample at a fixed wavelength. The data recorded in these conditions were corrected by software to remove the influence of the laser excitation frequency scanning. Fluorescence excitation spectra were obtained by scanning the laser excitation wavelength. In holeburning spectra, the broad band fluorescence was collected through a cut-off filter (all 2nuo> 645 nm). For site selection excitation spectra, the fluorescence was selectively recorded at a fixed wavelength behind the monochromator. Room temperature absorption
S.M. Arabei et aL / Chemical Physics Letters 232 (1995) 127-134
measurements have been performed with a conventional spectrofluorimeter (Philips, model PU 8720). The samples were placed inside a liquid helium bath cryostat with variable temperature facilities (SMC, FAir Liquide ). 3. Results and discussion
3.1. Absorption and fluorescence spectra at 300 and 4.2K Room temperature absorption and fluorescence spectra of hypericin in ethanol are shown in the Fig. la. The mirror symmetry rule for the fluorescence spectrum with respect to the absorption spectrum in the range 500-600 nm is fulfilled. This proves that the absorption observed in this spectral range is only due to the vibronic transitions of the $1 state. Nevertheless, a careful analysis shows that the vibrational frequencies measured in the excited S~ state (Avs,, in the absorption spectrum) are larger than the vibrational frequencies measured in the ground state (Avso, in the fluorescence spectrum). For instance, we measured a difference A Vs, - A Vso= 120 c m - 1for the high frequency vibronic band at 2600 cm-1 of hypericin in ethanol. This difference is also larger for the same bands in the absorption and fluorescence spectra of hypericin in the PVB film at 300 K (AVsl -Avso = 170 cm - I ). The 0-0 band of the fluorescence spectra of hypericin in ethanol is practically unshifted when the temperature is lowered to 4.2 K (see Fig. lb). However, the vibronic bands of the ground state are red-shifted by 2-4 nm, increasing the frequency intervals A Vso. A similar observation has been reported for the fluorescence of hypericin in ethanol at liquid nitrogen temperature [ 12]. The absorption spectrum (more precisely, the fluorescence excitation spectrum) is also red-shifted by 3.2 nm from room to liquid helium temperature (see Fig. lb). In this situation, we find that the vibrational frequencies are nearly equal in the ground state and in the first excited singlet state. For instance, the band at 578.2 nm in the fluorescence excitation spectrum assigned to a vibrational mode at Avs, =450 cm - l corresponds to a band at 610.5 nm in the fluorescence spectrum giving the same vibrational frequency A Vso= 450 c m - 1. At room temperature, the vibrational frequencies
129
are larger in the excited state than in the ground state. As also pointed out here, this difference disappears at low temperature. This observation perhaps reveals the occurrence of a possible dynamic equilibrium between different tautomers of hypericin (see further on) at room temperature. This equilibrium will be temperature dependent, resulting in the fact that one configuration only is expected to be populated at low temperature. A part of the high resolution site-selected fluorescence spectrum of hypericin in ethanol at 4.2 K is shown in Fig. 2a (curve ( 2 ) ) . The narrow dye laser excitation is located on the red edge of the inhomogeneous absorption band S 1~- So (during the registration of the fluorescence, the wavelength of the excitation laser was scanned from 596.7 to 593.3 nm). Despite the selective excitation with a narrow laser line, the vibronic band of the fluorescence spectrum around 703.2 nm (not shown in the figure) remains large and structureless. The average frequency interval for this band is large ( ~ 2608 c m - 1). A possible assignment for this band is to - O H stretching vibrations involved in hydrogen bonds, intramolecular or between guest and host. These vibrations are expected to be sensitive to local changes. We think that
i
'
570
'
~o
'
'
580
12bo
'
'
,,bo
590
'~,~-,
;% n m
Fig. 2. Fluorescence (a) and fluorescenceexcitation (b) spectra of hypericin in ethanol at 4.2 K. On top (a), curve ( 1): fluorescence using non-selective excitation with all argon laser lines; curve (2): fluorescenceunder selectiveexcitation with a monochromatic dye laser line (596.7-593.3 nm, Ave~c.~l cm-', Avr~ 1 cm-l).
130
S.M. Arabei et al. / Chemical Physics Letters 232 (1995) 127-134
this is a reason why, despite the selective laser excitation, the v i b r a t i o n a l f r e q u e n c i e s can be different resulting in a lack o f c o r r e l a t i o n b e t w e e n the S~ ( 0 ) So(v) intervals for centres excited t h r o u g h the channel S~ ( 0 ) ,-- So ( 0 ) . F o r this b a n d , the selective excitation is u n a b l e to e l i m i n a t e c o m p l e t e l y the i n h o m o geneous b r o a d e n i n g . I f the r e a s o n for the b r o a d e n i n g was s o m e v i b r o n i c congestion, we should observe this b a n d at low t e m p e r a t u r e at least partially resolved. We do not o b s e r v e a decrease in the f l u o r e s c e n c e intensity o f h y p e r i c i n u n d e r e x c i t a t i o n w i t h argon laser light at 4.2 K. In this case, p h o t o p h y s i c a l and p h o t o c h e m i c a l changes i n v o l v i n g h y p e r i c i n are in a stable e q u i l i b r i u m s i t u a t i o n r e a c h e d b e t w e e n direct a n d reverse l i g h t - i n d u c e d reactions. T h e fluorescence s p e c t r u m c o v e r i n g the range 5 9 5 . 0 - 6 8 5 . 0 n m for h y p e r i c i n u n d e r argon laser i r r a d i a t i o n at 4.2 K is s h o w n in Fig. 2a ( c u r v e ( 1 ) ). In this figure, we c o m pare the b r o a d b a n d fluorescence w i t h the site-selected fluorescence: as o b s e r v e d , the intensity distrib u t i o n a m o n g the b r o a d b a n d s is similar to the intensity d i s t r i b u t i o n a m o n g the fine lines o f the resolved spectrum. T h i s o b s e r v a t i o n p r o v e s that the m e t h o d used to r e c o r d the site-selected fluorescence, s c a n n i n g s i m u l t a n e o u s l y the e x c i t a t i o n f r e q u e n c y o v e r a small range, is efficient in e l i m i n a t i n g any influence o f a persistent h o l e - b u r n i n g m e c h a n i s m . T h e site-selected e x c i t a t i o n s p e c t r u m o f h y p e r i c i n in e t h a n o l s h o w n in Fig. 2b was o b t a i n e d r e c o r d i n g the fluorescence o v e r a n a r r o w spectral range a r o u n d 595 nm. T h e accessible spectral range is l i m i t e d to 0 750 c m - ' because o f the s c a n n i n g capabilities o f the dye used. All the vibrational frequencies m e a s u r e d for the g r o u n d state a n d the lowest e l e c t r o n i c state are p r e s e n t e d in T a b l e 1. F o r c o m p a r i s o n , we also s h o w the c o r r e s p o n d i n g r e s o n a n c e R a m a n d a t a p u b l i s h e d in the literature [ 17 ]. T h e m i r r o r s y m m e t r y b e t w e e n the fluorescence and the a b s o r p t i o n spectra is well p r e s e r v e d . We notice that the m o s t intense lines b e l o n g i n g to the 0 - 7 5 0 c m -~ range in the fluorescence s p e c t r u m (451 a n d 463 c m - i ) are not r e s o l v e d in the fluorescence excit a t i o n s p e c t r u m ( 4 5 0 c m - 1). S o m e d i s c r e p a n c i e s are o b s e r v e d b e t w e e n o u r results a n d the r e s o n a n c e Ram a n d a t a [ 17 ]: the difference can be as large as 13 cm-' for s o m e v i b r a t i o n lines ( f o r instance: A v s o = 1305 c m -1 a n d AVRR = 1292 cm -~, 6 = -- 13 c m - ' , o r also A v s o = 1647 c m - t a n d AVRR= 1636
Table 1 Vibrational frequencies (in cm -I ) of hypericin in ethanol in the ground state (AVso) and in the first excited singlet state (AVs,), compared to resonance Raman data (Avv.x) Avso
Avs~
143 226 298 301 351
384 401 451 463 490 520 580 608 627 649 685 705 752 903 929 948
145 220 296 305
-
318
-
340 376 400 422 436
-
1330
482 505 576 622 623 643
1471 1557 1601 1647 1705 1751 1761 1794 1829 1931 ~2608
627 -
690
693
748 -
920
-
-
1119 -
1351 1378 1402
-
450
1098
1136 1195 1257 1305
AVRR [ 17]
-
-
1245 1292 1313 1328 1370 1456 1499 1557 1596 1636 -
S.M. Arabei et al. / Chemical Physics Letters 232 (1995) 127-134
c m - ~, J = - 11 c m - ~). Also, some lines show different activities: for instance, the line at 1305 cm-1 in the site-selected fluorescence spectrum of hypericin has a large intensity compared to the corresponding line at 1292 c m - t which appears with a weak intensity in the resonance Raman spectrum. These differences are probably due to the fact that we compare two very different systems and experimental conditions: low temperature fluorescence data ofhypericin in ethanol glass on one hand, Raman data on a pure solid ofhypericin at room temperature on the other. A complete assignment together with a normal mode analysis of hypericin will be published in a forthcoming paper.
131 1.0
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~94
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3.2. Persistent spectral hole-burning
T 0 ~.,rl/tt
b Ethanol
08 0.8
Efficient persistent spectral hole-burning is observed for hypericin in ethanol glass as well as in PVB films. Some results are shown in Fig. 3 (top). In this work, we are interested in the stability of persistent holes and also in the photochemical hole-burning mechanism. We burnt deep holes in the inhomogeneous absorption band of hypericin in ethanol (Fig. 3a ( top ) ) or in the PVB film (Fig. 3 b ( t o p ) ) using ~ 1 mW cm -2 for 1000 s at 5.0 K. At the laser excitation frequency, the optical density decreased more than 70% in ethanol glass and more than 60% in the PVB film. In both matrices, a pseudo-phonon side hole is recorded with a maximum located at 18 cm-~ on the red-side from the narrow zero-phonon hole. A much weaker phonon side band is also detected on the blueside with a broad maximum at approximately 12 c m - i.
At the laser excitation frequency, a decrease of 40%-45% in the optical density at 5.0 K is performed with a burning time of 45 s with the following condilions: p~,th= 1 mW cm -2 at 2~,th =595.0 nm and P ~ W = l mW cm -2 at 2bPW=598.9 nm (Fig. 3a (middle)). Then, the burning process slows down and 65% of complete saturation is achieved after 10 min irradiation. The kinetics of hole growing at the early stage of burning is shown in Fig. 3b(middle). The hole-burning efficiency is higher in the ethanol glass than in the polymeric host. Despite the saturation of the zero-phonon hole, we notice that the growing of the pseudo-phonon side hole does not slow
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\ 0
2
4
6ts
.\,....~
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10
20
t, man
o 50
40 30 2C I0
o
oJ ,0
a
o/ s0
/ 30
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Fig. 3. (top) Hole-burning spectra of hypericin in ethanol (a) and in PVB (b) at 5.0 K (Wex~0.1 ~tW cm -~, 2nuo> 645 nm). Hole-burning conditions were: (a) 2b= 595.0 rim, tb= 1000 S, Wb~l mW cm -2, Tb~5.0 K, (b) 2b=598.9 nm, tb=1000 S, Wb~ 1 mW cm -2, Tb~ 5.0 K. (middle) Relative decrease of the absorption of hypericin in ethanol at 595.0 nm (a) and (b) and in PVB at 598.9 nm (a) at 5.0 K versus burning time ( Wb~ 1 mW cm-2). (bottom) Hole width versus temperature for hypericin in ethanol and in PVB.
down. This is understandable because this pseudoband is due to the burning of centres located on the red-side with respect to the laser excitation frequency
132
S.M. Arabei et al. /Chemical Physics Letters 232 (1995) 127-134
and which are excited through their phonon side band [20 ]. As the absorption in the phonon wing is weaker (see Fig. 3 (top)), the burning kinetic will be slower than for the zero-phonon hole. The hole-burning quantum efficiency is calculated according to the following equation [ 21 ]:
~hb=
[d( - ALl)/dt]t=o AOJh/Ao)i 103 Io( 1 _ 10-AO)~p '
where A is the absorption, Cp the molar extinction coefficient, Io the incident laser intensity on the sample, and AWh and Ao~i the homogeneous and inhomogeneous bandwidths, respectively. Using the data shown in Fig. 3b(middle), we estimate 4~hb~ 1.1×10 -2 which represents quite a high value. For comparison, the following values have been reported in the literature: for instance, for quinizarin in EtOH:MeOH glasses, q~hb~ 10 -4 [22], for some porphyrins, q~hb~ 10- 3 [ 21 ]. Higher hole-burning quantum yields have been evaluated only for twocolour photon gated hole-burning processes. As an example of an efficient two-photon gated spectral hole-burning system, we can mention the case ofperylene in boric acid glass for which a value q~hb~ 10has been estimated [23 ]. In Fig. 3 (bottom), we present a qualitative result showing the increase in the hole width with temperature for hypericin in ethanol and in PVB. For these experiments, the burning time has been changed from 1 to 5 min and the burning power from 1 to 10 mW cm-2 when the temperature was increased. The solid line results from an interpolation between the experimental points using a polynomial expression: it is presented here only for the sake of clarity of the figure. A precise fit of these data will have no simple meaning because hole widths were measured on nearly saturated holes. In these conditions, we cannot get the change in the homogeneous width with temperature. Nevertheless, as shown in the figure, the holes are broader in ethanol glass than in the polymeric host, even at 4.2 K; they also widen more rapidly. The fact that the holes are broader in ethanol than in a polymeric host has been reported several times in the literature, for different guest molecules, independently of the hole-burning mechanism, intramolecular or intermolecular photochemical hole-burning or non-photochemical hole-burning [ 24-26 ]. The
influence of hydrogen bonding between hypericin and the alcoholic host should be investigated, for instance, using deuterated ethanol as the host. This has not been done, but from the literature [25], it appears that proton tunnelling between hydrogen bonds, in the host or between guest and host, do not seem to be responsible for dephasing (and then for the hole width) as proved by the fact that no change is observed in the hole width when normal ethanol is replaced by deuterated ethanol in the case of resorufin. Conversely, deuteration has a drastic effect on the hole-burning efficiency which is strongly decreased in the deuterated host. From a different approach, differences in the hole widths between ethanol and polymeric hosts have been assigned to differences in the coupling constants between guest and host [ 27 ]. 3.3. Hypericin photoproduct
The presence of several hydroxyl groups at the periphery of the hypericin molecule suggests that a similar mechanism as the one suspected to explain the photochemical hole-burning of quinizarin in alcoholic solvents can also be assumed in this case. In the literature, two possible photo-rearrangements are discussed for quinizarin: the intramolecular proton transfer of one or two protons (tautomerization) and the occurrence of hydrogen bonding with the host (conformational changes) [ 22,28-31 ]. For these two cases, photoinduced changes in the hydrogen bonds of quinizarin involving one or two hydroxyl groups have been observed. In this work, we have made a systematic study of the changes in the excitation spectra (which is proportional to the absorption profile) of hypericin in ethanol with the increase in burning time, at a fixed frequency arbitrarily chosen at 595.0 nm in our experiment. We observed that the increase in the zerophonon hole depth and the pseudo-phonon hole depth induces the growth of a new absorption band on the red-side, around 600-603 nm (see Fig. 4). The superposition of all the fluorescence excitation spectra we have clearly shows an isobestic point at 598.5 nm. This point proves the existence of a spectral form of a photoproduct of hypericin which should result from a unique photoinduced change. When several spectral holes are burnt in the blue spectral range 594.3595.1 nm (Fig. 4, curve (4)), a corresponding blue-
133
S.M. Arabei et al. / Chemical Physics Letters 232 (1995) 127-134 i M .x
"-... \ "...\ ....\
%N o.
o
"n °-n-'°
I_ 3
Fig. 4. Fluorescence excitation spectra of hyperiein in ethanol at 5.0 K ( W ~ 0 . 1 ~tW cm -2, ,taro> 645 nm) after burning a hole at 2b=595.0 nm with Wb~l mWcm-2 and tb=0 S (1), tb=225 S ( 2 ), tb= 1600 S (3). The spectrum (4) has been recorded after burning several holes with the followingconditions: 2b= 595.0594.4 nm, tb=900 S, Wb~ 1 mW cm -2. shift is also observed in the absorption band of the photoproduct. The fluorescence excitation spectrum o f the photoproduct has two maxima: the first one is shifted to the red by 227 cm -~ from the position of the zero-phonon hole; the second one is located, not very clearly, in a 150-200 c m - 1 area, also on the redside. The existence o f a broad band corresponding to the photoproduct proves that the main photochemical reaction responsible for the hole-burning mechanism must be a tautomerization reaction. The reason is as follows. A careful analysis shows that all the possible intramolecular transfers of one or two protons of hypericin are accompanied by drastic changes in the conjugated electronic n system. In the insert of Fig. 4, we have drawn the four different conjugated electronic n systems of hypericin associated with possible tautomeric changes. In total, sixteen different configurations should be drawn, but most of them are equivalent and should correspond approximately to the same energies. These changes explain how different photoproducts absorbing at different frequencies can be produced. This situation differs from that of quinizarin because similar drastic changes in its conjugated system are not possible and a photoproduct cannot be definitely identified. As pointed out previously, these spectral forms probably exist also in dynamic equilibrium at room temperature. Nevertheless, we must add that the spectral forms ofhypericin located on the low energy side and which
appear during the photochemical hole-burning process are not unique. Our experiences show that the hole burnt at 595.0 nm is refilled partly if another hole is burnt on its blue-side (594.8 nm). This second hole also decreases when a new hole is burnt at higher energy (594.6 n m ) , and so on .... Fig. 5 describes these experiments: successive holes are labelled by numbers from 1 to 7 according to their chronological order. A spectral analysis of the complete hole shape shows that there are also other forms of photoproducts which are located on both sides of the burnt hole (the average spectral distribution for such photoproducts is estimated to be extended over -k-_7 cm-~ around the burning wavelength. This feature is characteristic of non-photochemical holeburning mechanisms thought to be only due to some
1VA: ~ :
---
V
J
--.
.
ii
ii~t
413,211
,7',6',s i i 594
I'~lth~ 595
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Fig. 5. Successive hole-burning in the absorption hand of hyper-
icin in ethanol at 5.0 K (tb=900 s, Wb~ I mW cm-2).
134
S.M. Arabei et al. /Chemical Physics Letters 232 (1995) 12 7-134
rearrangements in the host cage around the guest molecule. Nevertheless, this is not a definite proof because hypericin has the possibility of forming intermolecular hydrogen bonds with the host. Possible laser-induced changes among these hydrogen bonds would be a photochemical mechanism which would not involve a modification of the electronic conjugated system and then no drastic energy shifts.
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[ 13] P. Jardon and R. Gautron, J. Chim. Phys. 86 (1989) 2173. [14] A. Michaeli, A. Regev, Y. Mazur, J. Feitelson and H. Levanon, J. Phys. Chem. 97 (1993) 9154. [ 15 ] T. Yamazaki, N. Ohta, I. Yamazaki and P.-S. Song, J. Phys. Chem. 97 (1993) 7870. [16] E.B. Walker, T.Y. Lee and P.-S. Song, Biochim. Biophys. Acta 587 (1979) 129. [17]L.N. Raser, S.V. Kolaczkowski and T.M. Cotton, Photochem. Photobiol. 56 (1992) 157. [ 18 ] H. Pschierer, J. Friedrich, H. Falk and W. Schmitzberger, J. Phys. Chem. 97 (1993) 6902. [ 19 ] H. Brockman, K. Kluge and M. Muxfeldt, Chem. Ber. 90 (1957) 2302. [20] I.J. Lee, J.M. Hayes and G.J. Small, J. Chem. Phys. 91 (1989) 3463. [21 ] K. Horie, M. Ikemoto, T. Suzuki, S. Machida, T. Yamashita and N. Murase, Chem. Phys. Letters 195 (1992) 563. [22] Y. Iino, T. Tani, M. Sakuda, H. Nakahara and K. Fukuda, Chem. Phys. Letters 140 (1987) 76. [23]E.I. Alshits, B.M. Kharlamov and R.I. Personov, Opt. Spectry. 65 (1989) 326. [ 24 ] H.P.H. Thijssen, A.I.M. Dicker and S. V~51ker,Chem. Phys. Letters 92 (1982) 7; H.P.H. Thijssen, R. Van der Berg and S. V61ker, Chem. Phys. Letters 97 (1983) 295. [25] M. Berg, C.A. Walsh, L.R. Narasimhan, K.A. Littau and M.D. Fayer, J. Chem. Phys. 88 (1988) 1564. [26] W. Breinl, J. Freidrich and D. Haarer, Phys. Rev. B 34 (1986) 7271. [27] H.P.H. Thijssen, R. Van den Berg and S. V61ker, Chem. Phys. Letters 120 (1985) 503. [28] F. Graf, H.K. Hong, A. Nazzal and D. Haarer, Chem. Phys. Letters 59 (1978) 217. [29] J. Friedrich and D. Haarer, J. Chem. Phys. 76 (1982) 61. [30] T. Tani, Y. Sahakibara, Japan J. Appl. Phys. 31 part 1 (1992) 703. [31 ] T. Tani and A. Itami, Mol. Cryst. Liquid Cryst. 216 (1992) 247.