Assignment and anharmonicity analysis of overtone and combination bands observed in the resonance Raman spectra of carotenoids

Spectrochimica Acta.

Vol.50A,No.819, pp.

1467-1473, 1994 Copyright @ 1994 Elsevier Science Ltd Printed in Great Britain. All rights resewed

Pergamon 9%4-l3539(94)FxMt57-H

OS&t-8539/94$7.00+ 0.00

Assignment and anharmonicity analysis of overtone and combination bands observed in the resonance Raman spectra of carotenoids HIROMI OKAMOTO,YAEKO SEKIMOTOand MITSUOTASUMI Department

of Chemistry and Research Centre for Spectrochemistry, School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Received

7 December

1993; accepted 4 January 1994)

resonance Raman spectra of all-trun.r carotenoids have been observed in the region of 5000-500 cm-’ for samples in glassy solution at 77 K and in the in vivo state at room temperature. Prominent bands in the wavenumber region higher than 2000 cm-’ are assigned to either overtones or combinations of three modes due to skeletal stretches and the CHJ in-plane rock. From the wavenumbers of the observed Raman bands, anharmonicity constants for these three modes (including cross-term constants) are obtained. It is found that, for each carotenoid studied, the cross-term anharmonicity constant between the C=C and C-C stretches is significantly larger than the other anharmonicity constants. Abstract-The

1. INr~00~0r10N anharmonicity is important for discussing vibrational relaxation, vibrational dynamics and reaction coordinates or potential surfaces in elementary reactions. A number of experimental and theoretical studies on vibrational anharmonicities have been conducted for diatomic molecules and relatively small polyatomic molecules, especially for the X-H stretches (X=C, N, 0, etc.) and large-amplitude vibrations [l, 21. Anharmonicities of such molecular vibrations are large in general, and consequently overtone and combination bands can be observed by direct one-photon absorption spectroscopy. Recent developments in laser spectroscopy of molecules in free jets, such as stimulated emission pumping spectroscopy [3, 41 for example, have extended the subjects of research to vibrational modes with smaller anharmonicities. On the other hand, anharmonicity analyses for vibrations other than the X-H stretches of large molecules are more difficult. Large molecules are generally not suitable for free-jet experiments. There are few large molecules for which overtones and combinations of skeletal vibrations are clearly observed in the condensed phase. Carotenoids give a large number of overtone and combination bands due to vibrations having totally symmetric character (in-phase C=C and C-C stretches and CH3 rock) in their resonance Raman spectra observed in the solid state and in solution [5-71. Anharmonicities of these vibrations can be determined if the Raman shifts of overtone and combination bands are accurately measured. Such data may provide fundamental information on anharmonic coupling in large molecules. There is another motivation for undertaking accurate determination of vibrational anharmonicities of carotenoids. Recently, HAYASHI et al. [8] have reported picosecond time-resolved anti-Stokes resonance Raman spectra from vibrationally excited carotenoids in vim and in vitro (in toluene and in benzene). From picosecond anti-Stokes Raman intensity changes, they have found that electronically excited carotenoid molecules non-radiatively relax to vibrationally excited states of the ground electronic state within about 10 ps. The positions of the observed anti-Stokes Raman bands are shifted to slightly lower wavenumbers (in Raman shift) in comparison with the corresponding 1-O vibrational transitions in the ground electronic state. This observation indicates that the observed anti-Stokes Raman bands are attributable to vibrationally excited states of u > 1. In order to obtain more quantitative information on the relaxation process from the excited electronic state to the ground state by utilizing the observed picosecond timeresolved anti-Stokes Raman spectra, it is necessary to determine energy spacings between excited vibrational levels in the ground electronic state. SAITOet al. [6] have reported an anharmonicity analysis of B-carotene in the solid state at 77 K. For the above-mentioned purpose, however, it is necessary to extend this kind of

VIBRATIONAL

1467

H.

1468

OKAMOTO et

al.

study to other carotenoids in solution (and in uioo as well, if possible) with higher accuracy. In this paper, we report resonance Raman spectra for several carotenoids in glassy solution at 77 K and in the in oiuo state at room temperature in the region of 5000-500 cm-‘, and anharmonicity analyses based on the observed results.

2. EXPERIMENTAL canthaxanthin, @-apo-8’-carotenal, ethyl-p-apo-8’Carotenoids studied were b-carotene, carotenoate, rhodopin and spirilloxanthin (Fig. 1). The all-truns isomers of these carotenoids were used. The samples were purchased from Wako Pure Chemical Industries (B-carotene), Fluka Chemika-BioChemika (canthaxanthin) and Sigma Chemical Co. (@-apo-8’-carotenal and ethyl-/Iwas recrystallized from a benzene-methanol mixture. apo-8’-carotenoate). B-Carotene Canthaxanthin, /3-apo-8’-carotenal and ethyl-/3-apo-8’-carotenoate were recrystallized from a petroleum ether-ethanol mixture. Rhodopin and spirilloxanthin were extracted from Chromatium vinosum, a species of photosynthetic bacteria, and purified according to the procedure reported previously [9]. The purity of each carotenoid estimated by thin-layer chromatography and electronic absorption spectroscopy was higher than 95%. In order to clearly observe overtone and combination Raman bands, it was necessary to record the Raman spectra at low temperatures. Low-temperature Raman measurements were performed for the carotenoids dissolved in methylcyclohexane-isopentane (1: 1; abbreviated as MCP) or diethyl ether-isopentane-ethanol (5 : 5 : 2; EPA) and contained in a liquid nitrogen-cooled quartz Dewar cell at 77 K. The Raman spectra at 77 K of B-carotene, canthanxanthin, B-apo-8’-carotenal and ethyl-@-apo-8’-carotenoate were observed for both MCP and EPA solutions, whereas those of rhodopin and spirilloxanthin were obtained only for EPA solutions because of their low solubilities in MCP. The concentrations of the carotenoids in these solutions were in the range 1O-2-1O-6 mol dmm3. The in vivo sample was prepared as the RC-870 complex of Ch. vinosum dispersed in a sodium thioglycolate-Tris buffer solution according to the previously reported procedure [9]. This sample

OEt

Ethyl p- apo-8karotenoate \“” \

\

\\\\A\ Rhodopin

OMe \

OMe

\\\\\\\\A

Spirilloxanthin Fig. 1.

Chemical

formulae

of the carotenoids

studied.

Resonance Raman spectra of carotenoids

1469

contained spirilloxanthin as the major carotenoid component and a trace amount of rhodopin. The concentration of this sample was adjusted to give an optical density of =2 at 600 nm. The sample was contained in a rotating cell at room temperature. The Raman spectra were excited with lines from an Ar+ laser (NEC GLG 3300) in resonance with the 2-O electronic absorption bands of carotenoids at about 460 nm, in order to obtain sufficient overtone and combination Raman intensities [5, 71. The laser power incident on the sample was less than 20 mW. Raman scattered radiation was dispersed by a triple polychromator (SPEX 1877) and detected by an intensified photodiode array detector (EG&G PAR OMA III). The observed wavenumbers were calibrated by emission lines from a neon lamp over the entire spectral region. The use of this multichannel Raman spectrometer ensured higher accuracy in determining peak wavenumbers of the observed Raman bands, in comparison with the previous work by SAITOet al., which employed a scanning spectrometer [6].

3. RESULTSAND

DISCUSSION

Raman spectra for carotenoids in glassy solution at 77 K are shown in Figs 2-7. The Raman spectra in Figs 2-5 for /I-carotene, canthaxanthin, B-apo-8’carotenal and ethyl/?-apo-8’-carotenoate in MCP at 77 K are essentially identical to those observed from their EPA solutions at 77 K. Most of the prominent features in these spectra can be assigned to fundamental, combination and overtone bands arising from three vibrational modes having totally symmetric character. These three modes are primarily the C=C stretch (q, 1530-1500 cm-‘), C-C stretch (v~, 1160-1150 cm-‘) and CH3 in-plane rock (Ye, 1005-lOOOcm-‘) [lo]. Th e assignments of the observed Raman bands in the 5000-1000 cm-’ region are given in Table 1.

5000

I

I

I

I

4000

3000

2000

1000

RAMAN SHIFT km“

Fig. 2. Resonance Raman spectrum of B-carotene excited at 457.9 nm in MCP at 77 K.

IO

4doo

3&o

2doo

i&O

RAMAN SHIFT km” Fig. 3. Resonance Raman spectrum of canthaxanthin excited at 457.9 nm in MCP at 77 K.

1004

F

\ 1154

1514

RAMAN INTENSITY

1160

F1007

1531

RAMAN INTENSITY

*

1159

1528

3052

RAMAN INTENSITY

Resonance

Raman

spectra

1471

of carotenoids

! .‘c z

. IO

4000

Raman

spectrum

f

2000

3000

1000

RAMAN SHIFT km” Fig. 7. Resonance

of spirilloxanthin

excited

at 488.0 nm in EPA at 77 K.

where Uiand VPrefer, respectively, to the vibrational quantum number and the harmonic frequency of the ith mode, and xij represents the anharmonicity constants relating to the ith and jth modes. The parameters VPand xij can be determined by a least-squares fitting of the observed Raman shifts to energy spacings between vibrational levels expressed by Eqn (1). In this study, we performed the fitting based on Eqn (l), taking only the abovementioned three vibrational modes into consideration. The obtained parameters are listed in Table 2. Standard deviations for the parameters are less than 6.5 cm-’ for the harmonic frequencies and less than 2 cm- ’ for the anharmonicity constants. The frequencies of the Raman bands calculated by using Eqn (1) with the parameters listed in Table 2 are also shown in Table 1. The calculated frequencies agree with the observed values within f2 cm-‘, except for a few combination tones. These results indicate that the present three-mode model describes the Raman-active energy levels of the carotenoids under study satisfactorily. It should be borne in mind, however, that the parameters obtained may effectively contain contributions from other vibrational modes, including those having non-totally symmetric character. Table 1. Observed

and calculated

frequencies

BCS

(in cm-‘) of fundamental, bands of carotenoids

CXS

ALS

overtone

ATS

and combination

RDS

Raman

SXS

01

02

ujt

Obs.

Calc.

Obs.

Calc.

Obs.

Calc.

Obs.

Calc.

Obs.

Calc.

Obs.

Calc.

0 0 1 0 0 0 1 1 2 0 0 1 1 2 2 0 3 1

0 1 0 0 1 2 0 1 0 1 2 1 2 0 1 2 0 2

1 0 0 2 1 0 1 0 0 2 1 1 0 1 0 2 0 1

1005 1160 1525 2009 2164 2315 2528 2681 3048 3168 3319 3678 3831 4050 4197 4316 4564 4831

1005 1160 1526 2009 2163 2315 2529 2680 3048 3168 3318 3681 3830 4049 4196 4320 4565 4830

1007 1158 1521 2010 2163 2314 2527 2674 3037

1007 1158 1520 2010 2163 2314 2527 2673 3038 3168 3315 3677 3823 4043 4185 4312 4553 4824

1004 1159 1528 2006 2163 2317 2531 2683 3052

1004 1158 1527 2006 2161 2316 2530 2684 3053 3161 3319 3685 3939 4054 4206 4318 4576 4839

1007 1160 1531 2008 2169 2319 2535 2687 3057

1007 1160 1531 2008 2169 2319 2536 2687 3057 3179 3326 3691 3842 4059 4209 4331 4580

1004 1154 1514 2006 2156 2304 2516 2662 3024 3156 3308 3662 3802 4027 4161

1004 1154 1514 2005 2157 2305 2517 2660 3025 3157 3307 3662 3802 4026 4162 4306 4532 4803

1001 1150 1508 2000 2150 2297 2506 2650 3012 3147 3297 3646 3790 4013 4151

1001 1150 1508 2000 2149 2297 2508 2651 3012 3147 3297 3650 3792 4011 4148 4298 4512 4793

3315 3674 3822 4044 4185 4553 4824

3318 3683 3838 4053 4207 4576 4841

3325 3688 3842 4060 4209

t u,, u2 and o3 are the vibrational quantum numbers, respectively, for the vibrational modes $ BC, B-carotene; CX, canthaxanthin; AL, B-apo-8’-carotenal; AT, ethyl-/I-apo-8’-carotenoate; pin; SX, spirilloxanthin.

4510

Y,, v2 and vj. RD, rhodo-

H. OKAMOTOet al.

1472 Table 2. Harmonic Harmonic Carotenoidt

frequencies

Anharmonicity

v2

v3

1535.1

1167.1

1006.9

(4.3)

(1.8)

(5.2)

cx

1526.1

1165.2

1013.4

(2.3)

(2.4)

(4.3)

AL

1532.0

1161.2

1008.6

(2.5)

(2.5)

(4.5)

AT

1539.0

1163.0

1015.4

(4.6)

(4.5)

(6.4)

RD

1521.7

1161.5

1008.0

(2.6)

(3.0)

(3.2)

1514.8

1156.2

1003.5

(3.7)

(4.3)

(5.1)

sx

constants (in cm-‘) of carotenoids

frequency*

VI

BC

and anharmonicity

XII -2.3 (0.5) -1.3 (0.5) -1.2 (0.6) -1.3 (1.0) -1.6 (0.6) -1.9 (0.8)

x22

constantt

x33

-1.8

-0.2

(0.4)

x12 -6.2

(1.2)

-1.3

-2.1

(0.4)

-1.5

(0.8)

-0.2

(1.4)

-1.5

(0.9)

(0.8)

-0.5

(1.2)

(1.0) -1.6

(0.7)

(1.0)

-6.2

-1.9

(1.3)

(1.8)

-8.2

(1.1)

-1.4

-0.9

-2.6

(1.9)

-1.5

(0.7)

-5.5

(1.4)

-1.3

-1.9

(0.9)

(1.2)

-0.3

XI3

-1.0

(0.9)

(1.1)

-6.4

(1.5)

-0.1

(1.2)

(1.5)

x23 -1.4 (0.8) -3.0 (0.9) -1.1 (0.9) -1.4 (1.3) -1.0 (0.9) -0.6 (1.2)

t For abbreviations, see footnote to Table 1. $ Standard deviation of each parameter is shown in parentheses.

The absolute values of all the anharmonicity constants obtained are less than 1% of the harmonic frequencies. This result is consistent with that of a simple one-mode anharmonicity analysis on shorter conjugated polyene molecules with four to six conjugated double bonds [12]. The cross-term (coupling) anharmonicity constant between the C=C and C-C stretches is larger than the other anharmonicity constants in all the carotenoids studied. This result is possibly related to the fact that displacements of the carbon atoms make large contributions to both of these two modes, and may provide important information on discussing vibrational relaxation pathways involving these two modes. Although the vibrations are nearly harmonic, the energy spacings between adjacent levels gradually decrease with increasing u. As shown in Table 3, the energy spacings for the Y,, v2 and v3 of spirilloxanthin decrease by 3-4, 2-3 and l-2 cm-‘, respectively, with an increment of one in u. These results may be useful for analysing the low-frequency shifts of the picosecond time-resolved anti-Stokes Raman bands reported by HAYASHI et al. [8]. The Raman spectrum of the in uiuo sample is shown in Fig. 8. As is mentioned in the Experimental section, the major carotenoid contained in the sample (RC-870 complex) is spirilloxanthin. Since this measurement was done at room temperature, overtone and combination bands could be observed only up to 3500 cm-‘. The Raman shifts for the in uiuo sample agree with those for spirilloxanthin in glassy solution at 77 K within 3 cm-‘. This result indicates that the principal features of the Raman spectrum in Fig. 8 are due to spirilloxanthin, and that the anharmonicities for the in uiuo state at room temperature are similar to those in EPA at 77 K. Therefore, the anharmonicity constants in Table 2 may be used for analysing the Raman spectral data obtained from the in uiuo samples. Table3.

Observed and calculated spacings (in cm-‘) between vibrational levels (of-u”) of spirilloxanthin VI

v2

adjacent

v3

Quantum numbers “1-“1)

Obs.

Calc.

Obs.

Calc.

Obs.

Calc.

1-o 2-l 3-2 4-3 5-4

1508 1504 1498 -

1508 1504 1501 1497 1494

1150 1147 -

1150 1147 1145 1143 1140

1001 999 -

1001 999 997 995 994

1473

Resonance Raman spectra of carotenoids

I

I

I

1

3000

2000

1000

RAMAN SHIFT /cm-’ Fig. 8. Resonance Raman spectrum of RC-B870 complex of Ch. oinosum excited at 488.0 nm at room temperature.

4. CONCLUSIONS

The following points have been clarified in the present study. (1) The bands observed in the 5000-2OOOcm-’ region of the resonance Raman spectra of the six carotenoids, both in glassy solution at low temperature and the in oiuo state at room temperature, are assignable as either overtones or combinations of only three strongly Raman-active modes, namely y1 (C=C stretch), v2 (C-C stretch) and v3 (CH3 in-plane rock). (2) According to the anharmonicity analyses based on Eqn (l), the cross-term anharmonicity constant between v, and v2 is significantly larger than the other anharmonicity constants for each carotenoid studied. (3) The energy spacings between adjacent vibrational levels for each of vl, v2 and v3 decrease by 3-4, 2-3 and l-2 cm-‘, respectively, with an increment of one in the corresponding vibrational quantum number.

REFERENCES [l] F. F. Grim, Ann. Reu. Phys. Chem. 35, 657 (1984). [2] M. Quack, Ann. Reo. Phys. Chem. 41,839 (1990). [3] C. E. Hamilton, J. L. Kinsey and R. W. Field, Ann. Reo. Phys. Chem.

37, 493(1986). [4] F. J. Northrup and T. J. Sears,Ann. Reu. Phys. Chem. 43, 127(1992). [S] F. Inagaki,M. Tasumiand T. Miyazawa,1. Molec. Spectrosc. SO, 286(1974). [6] S. Saito, M. Tasumiand C. H. Eugster,/. Ruman Specfrosc. 14, 299 (1983).

[7] H. Okamoto, S. Saito, H. Hamaguchi, M. Tasumi and C. H. Eugster, J. Raman Specrrosc. 15,331 (1984). [8] H. Hayashi, T. L. Brack, T. Noguchi, M. Tasumi and G. H. Atkinson, J. Phys. Chem. 95,6797 (1991). [9] H. Hayashi and S. Morita, J. Biochem. 88, 1251 (1980). lo] S. Saito and M. Tasumi, /. Ramun Specfrosc. 14, 310 (1983). 111 E. B. Wilson Jr., J. C. Decius and P. C. Cross, Molecular Vibrations. McGraw-Hill, New York (1955). 121 B. E. Kohler, C. Spangler and C. Westertield, J. Chem. Phys. 89, 5422 (1988).