J. Phys. Chem. 1982, 86,4004-4007
4004
Polarization Excitation Spectrum of Thymine Fluorescence in Neutral Aqueous Solution at -300 K. Evidence for an n7r* Transition James P. Morgant and Malcolm Danlels’ Chemistty Depertment, Oregon State University, Corvallis. Oregon 97331 (Received: February IO, 1982; In Final Form: April 6, 1982)
The fluorescence polarization excitation spectrum has been measured for thymine in aqueous solution at -300 K from 240 to 296 nm. The anisotropy is found to be essentially constant across most of the absorption band but shows a pronounced depolarization at the red edge of the band. A simple model is presented accounting for the depolarization in terms of a weak out-of-plane absorption located under the red edge. It is suggested that this is a “hidden” na* transition.
l b is the corresponding background (buffer, impurity The heterocyclic bases which encode the genetically significant information of DNA contain both bonding ( r ) emission, etc.). The fluorescence from thymine is clearly strongly polarized, in agreement with previous results. On and nonbonding (n) pairs of electrons, and low-energy UV excitation at 296 nm, the results shown in Figure 2 are excitation should thus occur by both r r * and na* transitions. The former should give rise to strong absorptions obtained. Despite the overlap of the Raman scattering, (willator strength f > 0.1) polarized in the molecular plane a major portion of the fluorescence is still clearly resolved while the latter should give rise to weakly allowed tranand there is considerable fluorescence intensity over the background. The noticeable change from exciting at 260 polarized perpendicular to the molecular sitions nm is that the emission is now extensively depolarized, in plane and lying at lower energies than the m* transitions. agreement with earlier unpublished results from OSU. While the AT*transitions are easily recognized and have Polarized excitation spectra have been obtained on the been characterized by a variety of approaches, no evidence OSU instrument by observing the emission at 350 nm and has so far been obtained for the low-lying n r * states. Some time ago we found that the very weak room-temscanning excitation wavelengths from 248 to 296 nm, with perature fluorescence from thymine in aqueous solution appropriate orientations of polarizers. The resultant emission anisotropy, defined as R = (Ill is strongly polarized; this has been confirmed,2 and, as - II)/(Ili + 21,) and corrected for instrumental polarizathe polarization at room temperature is identical with that tion artifacts by method c of Azumi and M ~ G l y n n is ,~ observed at -120 “C, we concluded3 that there was no shown in relation to the absorption spectrum of thymine evidence for rotational depolarization occurring before ) Figure 3. It can be seen that the emission is of constant emission. This is not surprising as the lifetime (7 = 4 ~ ~ in and high polarization over most of the absorption band is probably subpicosecond. Thus, the room-temperature but decreases rapidly on the leading edge as the M) energy polarization appears to be “intrinsic”. We now report the variation of the polarization of emission with the waveis approached. That the falloff is not due to change of length of polarized excitation (“fluorescence polarization emission geometry with decreased absorption intensity is excitation spectrum”) and show how the results may be shown by separate counting experiments at 240 nm where the extinction coefficient is the same as at 284 nm (much interpreted in terms of a “hidden” low-energy nr* tranlonger counting is needed here as the lamp intensity is very sition. Results reported here have been obtained by using two low). An emission anisotropy of +0.28 is found, close to different experimental arrangements, one at Oregon State the value at A- and quite different from the +0.18 at 284 nm. University (OSU), the other at the Laboratoire Curie, Universite Pierre et Marie Curie (Paris VI). Both employ We have considered two possible interpretations of this behavior. The first is that the system exhibits dual xenon arc excitation through double monochromators, the fluorescences originating in different excited states of the emission being detected by single-photon counting into a molecule, analogous to the ‘La and ‘Lb states of aromatic multichannel analyzer. The OSU apparatus has been described earlier,3 and the Laboratoire Curie apparatus is systems. For this to be possible the rate of emission must be comparable to internal conversion; however, emission a slightly modified version of that described by Vigny and occurs within 1 ps at 300 K,6 so this is perhaps not imD~quesne.~ In the present context the major difference possible. However, we feel that the weight of available between the arrangements is that the OSU apparatus uses evidence is against this. Thus, within the ranges available f i b polarizers (Polacoat PL-40 for excitation and Polaroid to our measurement the depolarization of anisotropy is HNP’B for emission) whereas the Laboratoire Curie apapparent across the entire emission band. Exciting at the paratus uses large calcite prisms. All measurements have been made on dilute M) solutions to avoid possible 0-0level, of course, limits emission measurements to X > 350 nm, but we have also excited at wavelengths between aggregation effects, and the cuvette position was adjusted in the manner described by Vigny and Duquesne4 to 260 and 296 nm, and the essential point is that we never compensate for moderate optical densities, so that the observe a significant variation of depolarization of aneffective optical density at 261 nm was -0.2. Figure 1 shows the scattering (Rayleigh and Raman) and fluores(1)M. Daniels and J. P. Morgan, Photochem. Photobiol., 27,73(1978). cence observed on the Laboratoire Curie apparatus when (2)P.R.Callis, Chem. Phys. Lett., 61,563 (1979). exciting at 260 nm with nonpolarized excitation; Figure (3)J. P.Morgan and M. Daniels, Chem. Phys. Lett., 67,533 (1979).
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’Present address: Biochemistry Department, Colorado State University, Fort Collins, CO 80523.
(4)P.Vigny and M. Duquesne, Photochem. Photobiol., 20,15(1974). (5)T.Azumi and S. P. McGlynn, J. Chem. Phys., 37, 2413 (1962). (6)M.Daniels and W. Hauswirth, Science, 171,675 (1971).
0022-3654f82/2086-4004$0I.25f0 0 1982 American Chemical Society
Excitation Spectrum of Thymine Fluorescence
The Journal of Physical Chemistry, Voi. 86, No. 20, 1982 4005
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length (corrected, of course, for the variation of exciting intensity with wavelength) essentially superimposes on the absorption spectrum. Though the evidence is not as direct as one would wish, it seems that the emission is homogeneous, i.e., occurs from a single state regardless of exciting wavelength. The second interpretation is that we are exciting more than one absorption moment in the wavelength region of concern and that these moments have different orientations with respect to the emitting moment. Consequently, the polarization of the emission depends on the wavelength variation of the overlap of the absorption moments. Phenomenologically we can regard the constancy and high polarization over the wavelength range from 248 to 276 nm as evidence that in this range we are exciting only one transition, whereas from 276 to 294 nm we see evidence of increasing incidence of a depolarizing transition. The simplest model would thus involve only two states in absorption and one in emission, and this model may be developed in a semiquantitative manner to extract characteristics of the depolarizing transition. All theoretical calculations, for example ref 8, indicate that the first absorption band of thymine with an oscillator strength of 0.22 corresponds to a single allowed aa* transition. Thymine has C, symmetry, and thus transitions can be either in plane or out of plane. The known aa* transition is polarized in plane: and we proceed on the assumption that the new depolarizing absorption is out of plane. When the geometry is thus fixed, the relative contribution of in-plane and out-of-plane moments to total absorption (the absorption anisotropy) can be calculated from the measured emission anisotropy. From the definition of anisotropy, a measured value, R , is the weighted contribution of the intrinsic anisotropies of the contributing components i
where R1 is the intrinsic anisotropy of component 1 and fl is its fractional contribution, etc. If we denote the emission due to in-plane absorption as f!p and that following out-of-plane absorption as fop, and If the efficiency . . *.*q of emission is the same for absorption by in-plane or I I . I 300 350 400 450 out-of-plane moments, then the extinction coefficients for WAVELENGTH, nm these two moments are related as (a) Scattering and emission from thymine (lo4 M) at pH 7.3.
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Figwe 1. Nonpdarired excitation at 260 nm: (0)emission polarizer vertical (V); (+) emission polarizer horizontal (H). Rayleigh scattering, 50K counts/channel full scale for V, at 260 nm; Rayieigh scattering, 5K counts/channel full scale for H, at 260 nm; Raman scattering, 10K counts/channel full scale for V, at 285 nm; Raman scattering, 5K coonts/channei full scale for H, 285 nm; fluorescence scattering, 5K countslchannel full scale for V and H. (b) Control measurements on buffer solution pH 7.3. wlthout thymine. Rayielgh scattering, 50K counts/channel full scale for V, at 260 nm; Rayieigh scattering, 10K counts/channel full scale for H, at 260 nm; Raman scatterlng, 50K counts/channei full scale for V, at 285 nm; Raman scattering, 5K counts/channei full scale for H, at 285 nm; fluorescence background, 5K countslchannel full scale for V and H.
isotropy with emission wavelength which would be expected if we were observing mixed emissions whose relative populations depend on exciting wavelength. In agreement with this, Vigny and Duquesne? who have been able to quantitatively take into account the extent of Raman scattering when this overlaps the emission, find that the integrated emission yield as a function of exciting wave(7)P. and Vigny M. Duquesne in "Organic Molecular PhotophyeiaP, Vol. 3,J. B. Birka, Ed., Wiley, New York, 1976,p 167.
Intrinsic anisotropy of emission is relatedlo to the angle 0 between the absorption and emission moments by R = 1/5(3cos20 - 1); if the emission moment is exactly collinear with the in-plane absorption moment (corresponding to a aa* fluorescence), then R should attain a value of +2/5, whereas, if 0 = a/2, R should be -ll5. We have earlier' reported a value of +0.34 at ,A, and have discussed3 reasons why the limiting value of +0.40 is not attained. If the emission moment is regarded as being in plane and at a small angle to the in-plane absorption moment, then the out-of-plane absorption moment is still orthogonal to it and the ratio Eip/tOp can be calculated by using R,, = -0.20. If the emission moment is not restricted to being in plane but can assume any direction in space, then the angle 0,- between the out-of-plane absorption moment and the emission moment is related to the angle Bire between the in-plane absorption moment and the emission (8) W. Hug and I. Tinoco, J. Am. Chem. SOC.,95,2803 (1973). (9)R. F. Stewart and N. Davidson, J . Chem. Phys., 39, 255 (1963). (10)F. Perrin, Ann. Phys. (Paris), 12, 169 (1929);A. Jablonski, 2. Phys., 96,236 (1935).
4006
Morgan and Danlels
The Journal of Fhysical Chemistry, Vol. 86, No. 20, 1982
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Figure 2. (a) Scattering and emission from thymlne (lo4 M) at pH 7.3. Nonpolarlzed exchation at 296 nm: (0)emission polarizer vertical (V); (+) emission polarizer horizontal (H). Raylelgh scattering, 500K counts/channel full scale for V, at 296 nm; Raylelgh scattering, lOOK countslchannel full scale for H, at 296 nm; Raman scattering, 50K counts/channel full scale for V and H, at 328 nm; fluorescence scattering, 5K counts/channel full scale for V and H. (b) Control measurements on buffer solution pH 7.3, without thymine. Raylelgh scattering, 500K counts/channel full scale for V, at 296 nm; Rayleigh scattering, lOOK counts/channel full scale for H, at 296 nm; Raman scatterlng, 50K counts/channel full scale for V, at 328 nm; Raman scattering, 10K countskhannel full scale for H, at 328 nm; fluorescence background, 5K counts/channel full scale for V and H.
moment, and the angle Oi, moments by" f/2(3 COS2 8,-
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With Bi, = x/2, then R,, becomes -0.15. For generality this latter value has been used in eq 1to extract E,, from the isotropic (rotationally averaged) absorption spectrum of thymine. The results are shown in Figure 4. We find (11) P. Soleillet, Ann. Phys. (Paris),12,23 (1929).
(12)B.J. Cohen, H. Baba, and L. Goodman, J.Chem. Phys., 43,2902 (1965). (13) S. F. Mason, J. Chem. SOC.,1240 (1959). (14)F. Halverson and R. C. Hirt, J . Chem. Phys., 19, 711 (1951). (15)M. Kasha, Discuss.Faraday SOC.,9,14 (1950).
J. Phys. Chem. 1082, 86,4007-4011
idence for the location and characterization of the n?r*
states of all of the DNA bases by the entirely independent technique of phosphorescence excitation spectroscopy16 and believe that the conclusions presented here are substantially correct. This appears to be the first experimental (16)J.-P. Ballini and M. Daniels, in preparation.
4007
observation of a low-lyingn?r* transition in the DNA bases.
Acknowledgment. Thanks are due to Alie Brunissen (Paris) for experimental assistance, Gene Snyder for data treatment, and J.-P. Ballini (Paris) for providing polarization factors. NSF provided partial support through grant PCM77-18040.
Raman Spectroscopic Evidence for Heteroionic Vibrational Coupling in Ammonium Nitrate I I I t 0. J. Kearley and S. F. A. Kettle' School of Chemical Sciences, UnlversRy of East Anglla, Norwich NR4 7TJ, United K i n N m (Recelved: February 19, 1982; In Final Form: May 24, 1982)
Previously available single-crystalRaman data for ammonium nitrate I11 are reinterpreted in the light of recent crystallographicdata and the recognition of heteroionic vibrational coupling. Frequency data are presented for ammonium nitrate I11 isotopically substituted with 15N03and N1803-in the presence of protonated and deuterated ammonium ions. These data show that the spectra are interpretable in terms of a modified factor-group model and reveal the presence of short-range order of ammonium-ion orientations in phase 111.
Introduction Ammonium nitrate is known to occur in at least five phases at atmospheric pressure, of which three, IV,' 111,2 and 113have been well characterized structurally. Of these the room-temperature phase, phase IV,and phase 11, stable between 85 and 125 "C, have clearly related crystal structures. However, a major quantitative difference between them is that in phase II both ammonium and nitrate ions are orientationally disordered whereas in phase IV both are ordered. In phase I11 (stable between 32 and 85 "C), the nitrate ions are ordered but the ammonium ions disordered. Although the crystallographic work has shown that in both phase 11and phase 111the ionic disorder takes the form of ions occupying sites with two different orientations, each with a 50% probability, such studies do not readily distinguish between the limits of total disorder and the existence of small domains of local order. It has been recognized that vibrational spectra, which may be sensitive to short-range interactions, provide a method which may distinguish between these limih4 The present communication is concerned with the interpretation of part of the Raman spectra of ammonium nitrate I11 and its isotopomers and consequent comment on the disorder in this material. In a previous paper we have presented, and discussed in detail, the vibrational spectra of ammonium nitrate IV in the 1400-cm-l r e g i ~ n . Whereas ~ other spectral regions are relatively straightforward, admitting of a site- or factor-group interpretation, the spectra in the 1400-cm-' region are rather complicated, although ultimately interpretable in terms of the factor group. A detailed analysis of the effects of partial and complete isotopic substitution showed that this complexity originates in an interionic coupling between the 6(NH2)modes of the ammonium ion and the v(N0) modes of the nitrate, modes which have characteristic frequencies in this spectral region. The Part XXVII of the series Solid-state Studies. 0022-3654/82/2086-4007$01.25/0
techniques of isotopic substitution and dilution have proved generally useful as probes for investigating interionic and intermolecular vibrational coupling, providing small and controllable perturbations to this mixing. Their application to ammonium nitrate 116demonstrated that a factor-group model making explicit allowance for orientational disorder is needed to explain the spectra. The clear implication is that the disorder is such that there is no domain pattern, for, if such a pattern existed, a phase-IV-like factor-group approach would be expected to be applicable. In an ordered structure, heteroionic vibrational coupling occurs between modes of the same factor-group symmetry species, but for disordered structures the selection rules would be expected to be less restrictive. Nevertheless, as we have shown elsewhere! they are not totally permissive; in particular, we have explained the fact that there are just two symmetry-distinct spectra which may be obtained in the single-crystal Raman spectra of ammonium nitrate II.6 Single-crystal studies of ammonium nitrate I11 present some problems. This phase is not readily prepared directly; rather it is usually prepared from phase IV. Unfortunately, although the IV I11 order/disorder transition is between two orthorhombic structures, the crystallographic axes are not preserved. This effect alone would probably cause degradation of the crystallites during thermal cycling, but, in addition, the ca. 5% volume in-
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(1) s. B. Hendricks, E. Posnjak, and F. C. Kracek, J . Am. Chem. Soc., 54, 2766 (1932). (2)B. W.Lucas, M. Ahtee, and A. W. Hewat, Acta Crystallogr., Sect. B,36, 2005 (1980). (3)B. W.Lucas, M. Ahtee, and A. W. Hewat, Acta Crystallogr., Sect. B,35, 1038 (1979). (4)J. C . Bellows, P. N. Prasad, E. M. Monbera, and R. Kooehan, Chem. Phys. Lett., 54, 439 (1978). (5) G . J. Kearley, S. F. A. Kettle, and J. S. Ingman, J. Chem. Phys., 73, 2129 (1980). (6) G . J. Kearley and S. F. A. Kettle, J. Crystallogr. Spectrosc. Res., 12, 79 (1982).
0 1982 American Chemical Society
