J. Phys. Chem. 1987, 91, 819-822 of coupling and intensity is characteristic of CCH rocks associated with cis bonds.15 What are the implications of these results for the structure of the retinal chromophore in BR548? The observation that both the CI3=Cl4 and C=N bonds in BR548 are in the cis configuration implies that the thermal BR568 BR548 transition involves a concerted “bicycle pedal” isomerization as initially proposed by Orlandi and S ~ h u l t e n . ~The ~ formation of the 13,15-di-cis structure in the dark also shows that the protein preferentially binds the di-cis chromophore since the all-trans geometry is the lowest energy form of the chromophore in solution. However, once the effects of CI3=CI4 and C=N isomerization are accounted for, the vibrational structure of BR548 is very similar to that of BR568indicating that differences in protein-chromophore interactions between BR568and BR548 are not pronounced. Recent solid-state N M R studies on BRS6*and BR548 have established the presence of a negative protein charge (or the minus end of a dipole) near Cs and a positive charge (or the plus end of a dipole) near C7.29aThere is also evidence for weak hydrogen bonding between the Schiff base and its protein c o u n t e r i ~ n ,and ~ ~ it - ~appears ~ that this weak hydrogen bond is the dominant mechanism in the “opsin shift” of BR.36-38 The similar I3C-chemical shifts for the two pigments support the idea that electrostatic protein-chromophore interactions in BR568and BR548 are not markedly different and suggest that steric differences in the retinal binding site may be responsible for stabilizing the BR548 chromophore. One of the most pronounced differences in the Raman spectra of and BRs48 is the large intensity of the CI4HHOOP mode in BR548. The intensity of this mode is probably induced by a local twist in the ground-state structure of the chromophore near
-
(33) Orlandi, G.; Schulten, K. Chem. Phys. Left.1979; 64, 370. (34) Hildebrandt, P.; Stockburger, M. Biochemistry 1984, 23, 5539. (35) Harbison, G. S.;Herzfeld, J.; Griffin, R. G. Biochemistry 1983, 22, 1.
(36) Nakanishi, K.; Balogh-Nair, V.;Arnaboldi, M.; Tsujimoto, K.; Honig, B. J . Am. Chem. SOC.1980, 102, 7945. (37) Lugtenburg, J.; Muradin-Szweykowska, M.; Harbison, G. S.;Smith, S. 0.;Heeremans, C.; Pardoen, J. A,; Herzfeld, J.; Griffin, R. G.; Mathies, R. A. J . Am. Chem. SOC.1986, 108, 3104. (38)Spudich. J. L.; McCain, D. A,; Nakanishi, K.; Okabe, M.; Shimizu, N.; Rodman, H.; Honig, B.; Bogomolni, R. A. Biophys. J . 1986, 49, 479.
819
C14, forcing the CI4Hhydrogen out of the plane of the conjugated T-system. This suggests that steric constraints in BRs48 do not allow the chromophore to fully relax following the BR568 BR548 conversion. This is consistent with the large upfield shift of the l3CI4N M R resonance which is attributed to steric interaction of the C14Hproton with the C-CH2 lysine protons across the cis C=N bond.20 Steric constraints near CI4 may also explain the reduced coupling between the C=N stretch and C15Hrock. In BR548 the C=N stretching mode shifts 5 cm-I upon 15-deuteriation, significantly less than the 11-cm-’ shift seen in BR56818 and the 12-15-cm-l shifts seen in the all-trans- and 1 3 - ~ i s - P S B . ~ ~ , ” In summary, the major differences between the resonance Raman spectra of BR568and BR548 can be directly explained by isomerization about the C I 3 = C l 4and C=N bonds. Characteristic vibrational frequencies and isotopic shifts previously described in all-trans- and 13-cis-retinal are observed in the spectra of the protein-bound PSB chromophores, demonstrating that the methods developed for determining chromophore structure in retinal model compounds are applicable for the retinal pigments. Thus, the results presented here should be transferable to the interpretation of the vibrational spectra of the other BR photocycle intermediates, as well as halorhodopsin and sensory rhodopsin.
-
Acknowledgment. We thank Anne Myers and Mark Braiman for insightful discussions and critical comments. Patrick Mulder, Chris Winkel, Jacques Courtin, Marcel van der Brugge, Albert Broek, Gerard Lam, and Ellen van den Berg synthesized the retinal isotopic derivatives employed in this work. This research was supported by the National Science Foundation (CHE-8 116042), the National Institutes of Health (EY-02051 and GM 27057), the Netherlands Foundation for Chemical Research (SON), and the Netherlands Organization for the Advancement of Pure Research (ZWO). R.M. is an N I H Research Career Development Awardee (EY-00219). Registry No. 13-cis-Retina1, 472-86-6.
Supplementary Material Available: The Cartesian and internal coordinates used in the calculations and a complete description of the force field will be found in Tables XIV and XV (7 pages). Ordering information is given on any current masthead page.
Polarized Absorption and Phosphorescence Spectra of Xanthone in Stretched Polyethylene Films Robert E. Connors,* Robert J. Sweeney, and Frank Cerio Department of Chemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609 (Received: July 21, 1986: I n Final Form: October 6, 1986)
Polarized absorption and phosphorescence spectra have been measured at several temperatures for xanthone embedded in stretched polyethylene films. On the basis of experimental results and molecular orbital calculations, a portion of the absorption spectrum has been reassigned. At 4.2 K the phosphorescencedata indicate that T,(TT*),of 3A1symmetry, obtains the major source of its radiative strength through a vibronic spin-orbit coupling mechanism involving the mixing of S,(nr*), of ‘A2 symmetry, and ‘ B 2 ( m * ) states through bl vibrations. Thermally activated phosphorescence is observed from T, which is separated from T! by 145 cm-’. Polarized phosphorescencespectra at 165 K support the view that T,(na*), of 3A2symmetry, derives its radiative activity through direct spin-orbit coupling to S 2 ( r r * ) ,of IAl symmetry.
Introduction Recent studies of the phosphorescence of xanthone in n-hexane1V2 have shown that the lowest triplet state, Tl(?r?r*),of 3A1symmetry and the second triplet state, T2(nT*),of 3A2symmetry are very (1) Connors, R. E.; Christian, W. R. J . Phys. Chem. 1982, 86, 1524. (2) Griesser, H . J.; Bramely, R. Chem. Phys. Left. 1981, 83, 287.
0022-3654/87/209 1-0819$01.50/0
closely spaced (A& = 29 cm-I). Griesser and Bramley3 have recorded and analyzed high-resolution phosphorescence spectra for the two emitting states in n-hexane. At 4.2 K they found that the T I emission could be resolved into two components, one arising from the TI, sublevel and the other from one or both sublevels (3) Griesser, H. J.; Bramely, R. Chem. Phys. 1982, 67, 373.
0 1987 American Chemical Society
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The Journal of Physical Chemistry, Vol. 91, No. 4, 1987
TI, and TI,. By lowering the temperature to 1.8 K, emission from is frozen out. TIZ,which is 1 1 cm-l higher in energy than At temperatures above 4.2 K, emission from T, becomes thermally activated and by 77 K Tz So phosphorescence virtually obscures emission from TI. The radiative decay of T I is dominated by z-sublevel emission with a symmetry allowed component and a much stronger vibronically induced component. By far the strongest mode appearing in the spectrum is the vibronic origin at 671 cm-l. These authors have considered the various spin-orbit and vibronic spin-orbit coupling routes that are the most likely candidates for inducing intensity into the phosphorescence of xanthone. The major source of T, radiative strength was assigned to direct spin-orbit coupling between TzZand S z ( m * ) of 'A, symmetry. Assigning the source of TI radiative intensity was less straight forward. Focusing on the dominant z-sublevel emission, Griesser and Bramley feel that the most plausible mechanisms responsible for the strong vibronically induced component are TI, e H,, SI H d a J Sz TI, Hdad T z ~ H,, SZ On the basis of energy gap considerations, the second mechanism involving vibronic coupling in the triplet manifold was favored. As the authors note, both of these possibilities have problems associated with them. First, the strong vibronic origin at 671 cm-l would have to be of a2 symmetry which is infrared forbidden for molecules classified under the Cz0 point group. However, the 671-cm-' mode appears with considerable intensity in the infrared s p e ~ t r u m . ~ Second, ,~ the polarized phosphorescence and absorption data of Pownall and Huber indicate that the vibronic bands are polarized negatively with respect to So SZs6They assign the main source of radiative intensity to S3 which is a KT* state of IB, symmetry. Relying on Pownall and Huber's polarization work, Charkrabarti and Hirota7 have offered as the most important vibronic spin-orbit coupling mechanism
Connors et al.
-
- +
+
-
-+
+-
-
Tiz SI Hdbi) 'B2 In rejecting this route, Griesser and Bramley observe that there is no obvious explanation why vibronic coupling of SI to S3should be more effective than coupling SI to Sz considering the larger energy gap and much smaller oscillator strength involved Lf(So-S2)/f(So+S3) = According to the assignments made by Pownall and Huber, S3is the only singlet state of B, symmetry above 225 nm that has significant intensity in the absorption spectrum. Griesser and Bramley also note the ambiguous nature of the polarization data. In light of subsequent work,1,8it is obvious that the polarized phosphorescence spectrum of xanthone in 3methylpentane at 77 K reported by Pownall and Huber contains contributions of comparable intensity from T l and T,. In an attempt to resolve some of the uncertainties and inconsistencies mentioned here, we have investigated the polarized absorption and phosphorescence properties of xanthone embedded in stretched polyethylene films. The films are well suited for measuring polarized spectra over a wide temperature range since they do not crack at very low temperatures or melt at high temperatures. Quantum mechanical calculations using the semiempirical M O SCF method HAM/39 were performed to assist in the assignment of the absorption spectrum. +
+
+
Experimental Section Xanthone was recrystallized twice from ethanol. Stretched polyethylene films were prepared following a published procedure.I0 Excitation was provided by radiation from a 150-W xenon (4) Klimenko, V . G.; Gastilovich, E. A. Russ. J . Phys. Chem. 1979, 53, 329. (5) Zwarich, R.; Binbrek, 0. S . Spectrochim. Acta., Parr A 1985, 41, 537. (6) Pownall, H. J.; Huber, J. R. J . Am. Chem. Soc. 1971, 93, 6429. (7) Chakrabarti, A.; Hirota, N. J . Phys. Chem. 1976, 80, 2966. (8) Pownall, H . J.; Connors, R. E.; Huber, J. R. Chem. Phys. Lett. 1973, 22, 403. (9) Asbrink, L.; Fridh, C . ;Lindholm, E. Chem. Phys. Lett. 1977,52,63. (10) Thulstrup, E. W.; Michl, J.; Eggers, J. H. J . Phys. Chem. 1970, 74, 3868.
250 300 350 WAVELENGTH ( n m ) Figure 1. Room temperature absorption spectrum of xanthone in unstretched polyethylene film.
200
arc lamp filtered through 10 cm of water and a Jarrell-Ash 82-410 m monochromator. Phosphorescence was dispersed by a Spex 1702 3 / 4 m monochromator and detected by an EM1 9659QB photomultiplier tube. After amplification with a Keithly Instruments 602 electrometer, the analog output of the photomultiplier tube was digitized by a Cyborg Corporation ISAAC 91A data acquisition and control interface system and an Apple IIe computer. Spectra were stored on disk for later manipulation and plotting. For recording polarized emission spectra, 3M Polacoat 105UV linear polarizers were introduced into the excitation and emission optical path. A Karl Lambrecht Corporation quartz scrambler was placed in front of the entrance slit of the monochromator to eliminate effects due to the unequal transmission of horizontally and vertically polarized light. The excitation polarizer was oriented in a fixed position to transmit vertically polarized light; the analyzer was rotated to monitor emission polarized either parallel or perpendicular to the vertical plane. Polyethylene films were mounted with the stretching direction vertical when exciting singlet state transitions polarized along the of the molecule and horizontal for short axis ( z ) long axis (j) polarized transitions. The degree of polarization was calculated -i Z1)/(Z1 i + ZL). An Air Products Displex 202 from P = (I closed-cycle refrigeration system was used to obtain emission spectra at 165 K. Samples were immersed in boiling liquid nitrogen and liquid helium to obtain spectra at 77 and 4.2 K, respectively. Absorption spectra were measured with a PerkinElmer 553 UV-visible spectrophotometer. Stretched film absorption spectra were obtained by mounting polarizers in the sample and reference compartments of the spectrophotometer. By rotating the film, spectra were recorded with the electric vector of the light beam parallel and perpendicular to the stretching direction. Base line corrections were made automatically by placing blank stretched films in the reference and sample beams during the background correction scan. HAM/3 CI calculations were performed on an AT&T 6300 personal computer with a program obtained from the Quantum Chemistry Program Exchange." Excitation energies were calculated by using 122 singly excited configurations. Results Figure 1 shows the room temperature absorption spectrum of xanthone in unstretched polyethylene film. This spectrum is quite similar to that obtained with hydrocarbon solvents such as 3methylpentane or n-hexane.6-'2 Prominent transitions are found at 338 (S,), 284 (S3), 259 (S4), and 238 nm (&). Polarized absorption spectra of xanthone embedded in stretched polyethylene film are presented in Figure 2. In this figure, parallel and perpendicular refer to the orientation of the linearly polarized light ( 1 1) Program QCMP 005, QCPE Bull. 1985, 5 , 33.
(12) Suga, K.; Kinoshita, M. Bull. Chem. SOC.Jpn. 1981, 54, 1651.
The Journal of Physical Chemistry, Vol. 91, No. 4 , 1987 821
Xanthone in Stretched Polyethylene Films
TABLE I: Observed and Calculated Singlet Excitation Energies for Xanthone energy, cm-' transition assignment obsd" obsdb IA2(nr*) 'Al 27050
f
calcd 27620 28 405 31 520 34610 37 123 40 406 40 630 40985
+-
'Al(***)
t
IA, IA,
29 585 35 210 38 610 -39 000 (tentative) 42015
IBZ(rr*) 'Bz(m*) +- IAl IA,(rr*) 'A, IB2(rr*) 'A, I B Z ( r r * )t 'A, I A l ( r a * )+- 'A, +-
+
29 450 35 280 38 750 42 670
-42 500 (tentative)
obsdb
calcd
polarization obsd" calcd
0.00 0.10 0.02 0.10 0.50
0.17 0.00 0.29 0.08 1.05 0.36 0.13
2
z
Y
Y
Y
Y
2
2
Y
Y Y z
z
In polyethylene film, this study. In 3-methylpe11tane.~ 1 .6
>-
Ic-.l
m z
W
W
0
z a m
!-
Z
H
LT 0
W
> c
m m
CI
U
a
J W
14I
0
0 c
c
3
250
300
3 0
WAVELENGTH ( n m ) Figure 2. Polarized absorption spectra of xanthone in stretched polyethylene film: (A) parallel orientation, (B) perpendicular orientation.
24000 26000 WAVENUMBER ( c m -1 ) Figure 4. Unpolarized phosphorescence spectrum of xanthone at 4.2 K in n-hexane (lower curve) and stretched polyethylene film (upper curve). The polyethylene spectrum has been displaced 120 cm-' toward higher energy for comparison. 20000
22000
W
u
z
6
[r
0 Ln
m
a
200
250 300 WAVELENGTH ( n m )
350
Figure 3. Reduced polarized absorption spectra: (A) long axis polarized
spectrum, (B) short axis polarized spectrum. vector with respect to the stretch direction. Using the TEMI3 method for reducing film spectra, and assuming that the long axis of the molecule partially aligns with the stretch direction, we have reduced the spectra to their y and,z components (Figure 3). The polarized spectra demonstrate clearly that the 338-nm transition is polarized parallel to the short axis of the molecule and that the 284-, 259-, and 238-nm transitions are polarized along the long molecular axis. Further, the reduced spectra suggest that there may be short axis polarized transitions (S, and S,) underlying the 259- and 238-nm transitions. Table I lists the experimental data and the results of the H A M / 3 calculations. Figure 4 shows the unpolarized phosphorescence spectrum of xanthone at 4.2 K in n-hexane and in stretched polyethylene film. Although the polymer environment produces considerable (13) Thulstrup, E.W.; Michl, J. J . Chem. Phys. 1980, 84, 82
O L : : : : : : : : : : : . : : 400
450
500
WAVELENGTH ( n m ) Figure 5. Phosphorescence and polarized phosphorescence spectra of xanthone in stretched polyethylene film at 4.2 K. The solid line corresponds to excitation into the 0-0 band of S, and the broken line to excitation into the 0-0 band of S,.
broadening of the spectral features, important bands, including the 0-0 region, the strong vibronic origin (671 cm-I), and the totally symmetric carbonyl stretch progression (1673 cm-I), are clearly resolved. Figure 5 shows phosphorescence and polarized phosphorescence film spectra of xanthone at 4.2 K. The solid line corresponds to excitation into the 0-0 band of the 338-nm transition (S,) and the broken line to excitation into the 0-0 band of the 284-nm transition (S3). With respect to excitation into S, the 0-0 band (25630 cm-I) of the phosphorescence and the 1670-cm-' totally symmetric carbonyl stretching vibration have positive and near-positive polarization, while the 300- and 670-cm-I vibrations have negative polarization. Excitation into
-
822
The Journal of Physical Chemistry, Vol, 91, No. 4, 1987 1
> I-
Connors et al.
-
forbidden n a* transition is not seen in the absorption spectrum of xanthone in a thin polyethylene film, it has been observed in 3-methylpentane solution by using a long path ceL6 The short axis polarized bands correspond to 'A, (m*) , A l transitions lA1. It is of particular and the long axis bands to 'B,(aa*) significance to note that our results and assignments differ from those of Pownall and Huber in the region below 270 nm. Whereas they have assigned the strong and very strong transitions appearing at 258 and 234 nm in 3-methylpentane (S, and S6in our notation) to z-polarized ]Al states, our experimental and calculated results clearly favor assigning these to y-polarized 'B2 states. Consequently, we find in the ultraviolet region, contrary to Pownall and Huber, that the total oscillator strength associated with transitions to 'B, states is considerably larger than for transitions to 'A, states. At 4.2 K where emission from T2 is absent the polarized phosphorescence data shows that the 0-0 band and totally symmetric carbonyl stretch are polarized parallel to S2(IA1). The remaining features, including the spectrally dominant 670-cm-' vibration, are polarized parallel to S3!lB2). Our results are similar to those obtained by Terada et al. using a modulated polarization method.I4 The major difference is that their emission remains y-polarized throughout the entire spectrum; however, the degree of polarization does drop considerably at the 0-0 and carbonyl stretch bands. It is obvious that the two mechanisms proposed by Griesser and Bramley are inconsistent with our polarized phosphorescence data. The vibronic spin-orbit coupling mechanism which we feel agrees most closely with our results is the one originally proposed by Charkrabarti and Hirota +
c1
cn
+
Z
W
h
Z
H
W
>
c1
I-
< _I
W IY
400
500
450
WAVELENGTH
(nm)
Figure 6. Phosphorescence and polarized phosphorescence spectra of xanthone in stretched polyethylene film at 77 K. Excitation into the 0 band of S,. Asterisks indicate emission originating from T,.
P
o
TI,
-. 5 0
.
.
.
400
.
.
.
450
WAVELENGTH
.
.
500
(nm)
Figure 7. Phosphorescence and polarized phosphorescence spectra of xanthone in stretched polyethylene film at 165 K. Excitation into the 0band of S2 A positively polarized progression in the carbonyl stretch originating from T, dominates the spectrum.
S3 produces the opposite behavior.
-
As the temperature is raised, population is brought into T, and T2 So emission characterized by a 1670-cm-I progression in the carbonyl stretch built onto the origin band at 25 775 tm-l begins to appear in the phosphorescence spectrum. At 77 K the phosphorescence is a combination of emission from Ti and T2, with T, more intense (Figure 6). The phosphorescence and polarized phosphorescence spectra at 165 K are presented in Figure 7. Emission from T, dominates the phosphorescence spectrum and has positive polarization with respect to excitation into S,. The negatively polarized components of T1emmission are clearly observed in the polarized phosphorescence spectrum. Subtracting the 0-0 band positions for TI and T, emission yields an energy gap of AET = 145 cm-'.
+
H,,
-
SI
+
H"(b1)
-
'B2
In light of the findings reported in this study, we believe that the objections to this assignment3 which were mentioned in the Introduction are not valid. With the reassignment of the absorption spectrum, it follows that transitions to IB2 states carry sufficient oscillator strength to serve as the primary source of radiative intensity. Additional support for this mechanism is given by the recent polarized infrared and Raman studies of Zwarich and Bir~brek.~They have assigned the 671-cm-I mode to a fundamental vibration of bl symmetry. The observation of z polarization at the 0 4 band and the totally symmetric carbonyl stretch mode suggests a mechanism involving direct electrostatic mixing between T I and T2.15 T1,
+
elect.
-
T,,
+
H,,
-
S2
From the weakness of the 04band relative to the 670-cm-I band it is seen that this mechanism is far less effective than the vibronic mechanism in conferring radiative strength to TI. The 165 K polarization data provide further experimental evidence in support of the previous assignment of the source of T2 radiative a ~ t i v i t y .Observation ~ of a strong 0-0 band and z polarization at the origin and totally symmetric carbonyl stretch mode is consistent with direct spin-orbit coupling between TZz and S,.
Discussion
Acknowledgment. The closed-cycle refrigeration system was acquired with funds provided by the National Science Foundation under Grant No. PRM-8208786. The WPI Office of Academic Computing provided the computer that was used for the molecular orbital calculations. Registry No. Polyethylene, 9002-88-4; xanthone, 90-47-1.
It is seen from Table I that a comparison of experimental data and theoretical results leads to a relatively straightforward assignment of the ultraviolet absorption spectrum. The calculation predicts that SI is of A, symmetry. Although this symmetry
(14) Terada, T.; Koyanagi, M.; Kanda, Y . Bull. Chem. SOC.Jpn. 1980, 53, 352. ( 1 5 ) Harrigan, E. T.; Hirota, N. Chem. Phys. Lett. 1974, 27, 405.
