Excitation Profiles of Astaxanthin
1137
Correlation between the Absorption Spectra and Resonance Raman Excitation Profiles of Astaxanthinla V. R. Salares,* R. Mendelsohn,lbP. R. Carey, and H. J. Bernstein Division of Chemistry and Division of 8iological Sciences, National Research Council of Canada, Ottawa, Canada K1A OR6 (ReceivedJanuary 5, 1976) Publication costs assisted by the National Research Council of Canada
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Resonance Raman excitation profiles in the region 4579-5287 8, are reported for the carotenoid molecule astaxanthin at -162 and 23 "C. At these temperatures the visible absorption spectra are structured and unstructured, respectively. The experimental data, simulated using a simple model, demonstrate that there is a correlation between the development of vibrational structure in the absorption spectrum and in the excitation profile. For astaxanthin the excitation profiles a t room temperature do not resolve the vibrational structure of the broad absorption band but do show that the 0-0 transition occurs near 18 850 cm-l.
tone-petroleum ether (bp 60-70 "C) gave a single band. The recrystallized material consists of only one isomer, and this Excitation profiles (variation of Raman intensity with exis believed to be the all-trans form.7 citing wavelength) have recently been suggested as a probe Absorption Spectra. Absorption spectra were measured of vibrational structure in absorption spectra.2-5This implies with a Cary 14 spectrophotometer. Low temperature spectra that vibrational structure which lies hidden in an absorption in EPA (diethyl ether-isopentane-ethanol, 5 5 2 by volume) band may be resolved by measuring Raman excitation profiles. were obtained using a dewar equipped with quartz windows. To date excitation profiles have been measured primarily for A 2-mm quartz cell which fitted inside the dewar cavity was molecules with structured absorption ~ p e c t r a . Thus, ~ ~ - ~it is cooled by flowing cold nitrogen gas. Temperatures were not clear if any correlation exists between the resolution obmonitored with a chromel-alumel thermocouple. tainable in the Raman excitation profiles and in the absorpRaman Spectra. Raman spectra were recorded using 90" tion spectrum. T o study this correlation, the carotenoid scattering geometry on a Jarrell-Ash Model 25-102 double molecule astaxanthin (3,3'-dihydroxy-P,P-carotene-4,4'-dione) monochromator equipped with an RCAX31034 photomul(I) was selected since its Raman excitation profiles could be tiplier tube and photon counting detection. A Spectra Physics 0 II 166 Ar+ laser provided nine excitation lines from 4579 to 5287 8;output a t 5287 8 was obtained by modifying the laser optics. Laser powers were kept between 4 and 9 mW. The vibrations of interest were scanned alternately with the referastaxanthin 0 ence lines at least three times. For room temperature measurements, the rotating cell techniques was used to minimizelocal heating. The solution of astaxanthin in chloroform (A,,, = 0.96) was first deoxyP-carotene genated and purged with nitrogen gas. Under these conditions, the absorbance and resonance Raman spectra were invariant obtained under conditions of both structured (low temperaindicating the absence of photochemical degradation or ture) and unstructured (room temperature) absorption isomerization. The intensities of the 1524- and 1157-cm-l spectra. A simple model, similar to that applied in previous vibrations of astaxanthin (Figure 1)were measured relative Raman studies,2b4 was used to analyze the intensity data and to that of a chloroform line a t 366 cm-l. semiquantitatively evaluate the temperature dependence. For low temperature measurements, an astaxanthin soluExperimental Section tion in EPA (A,,, = 1.40) was sealed in a glass capillary and mounted on an unsilvered, double-jacketed glass dewar.9 Solvents and Reagents. Spectrochemical grade chemicals Cooling was achieved by flowing a stream of cold nitrogen gas. were used when available. The intensities of the two fundamentals at 1524 and 1157 Astaxanthin. Astaxanthin was extracted from cleaned and cm-I and of the overtone at 3045 cm-l were measured relative ground lobster carapace with acetone at 5 "C. Purification and to the total area of solvent bands at 2808,2874,2938 and 2982 recrystallization from dichloromethane-petroleum ether (bp cm-l. Each set of measurements with the nine exciting lines 30-60 "C) by the method of Buchwald and Jencks6 yielded was obtained continuously. Intensity ratios with 5145-%,exred-violet platelets. The crystals or solutions in dichlorocitation at the beginning and at the end of the measurements methane were stored at -23 "C. were reproducible to f 5 % . The absorption spectra in different solvents were identical Treatment of Experimental Data. Intensities were meawith those reported earlier.6 Thin-layer chromatography on sured as band areas with a planimeter. An average value of silica gel 60F-254 (0.25 mm, Merck) developed with 20% acethree or more measurements on each line was taken and this was corrected for the wavelength dependence of the instru* Address correspondence t o this author a t the Division of Chemistry, National Research Council of Canada. ment response. The latter had previously been calibrated
Introduction
The Journal of Physical Chemistry, Vol. 80, No. 11. 1976
1130
Salares et al.
the excitation profiles. Simplification of the A term occurs because only one excited electronic state is assumed to be in resonance and because under resonance conditions the second term in eq 1becomes negligible. The depolarization ratios for the vibrations studied for astaxanthin were about 0.3, indicating that only one term in ) contributing to the observed the scattering tensor ( a z zwas intensity. In this instance eq 1 reduces to the following form:
= constant x f a 2
((Y,,)~
where
I6W
1200
14W
1000
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FREQUENCY S H I F T (crn")
Figure 1. Resonance Raman spectrum of astaxanthin in CHC13 at +23 " C , 4658-A excitation; 4 mW laser power, 6.5 cm-' spectral slit width, scan speed 0.5 cm-'/s; full scale sensitivity 3000 counts/s.
using a standard lamp. Since the reference lines were not adjacent to the astaxanthin vibrations, a X4 correction3 was also applied. The solutions were sufficiently dilute (8.5 X M in chloroform, 1.25 X M in EPA) that corrections due to self-absorption were considered to be neg1igible:'j
Theoretical The model used to calculate the excitation profile is based on the form of the scattering tensor derived by AlbrechtlO and co-workers. The upncomponent of the tensor is given by: (a,>,)gl,g,
=A
+
where
where the terms involving the dipole moment matrix elements are included in the constants. Several approximations are introduced to evaluate fa: (a) harmonic oscillator wave functions are assumed for the g and e states, (b) it is assumed that no change in force constant occurs in the upper state, and (c) it is assumed that the potential energy curves are displaced from each other horizontally by a parameter x such that x =
(Vb.,1,7r/h)1/2Q
(4)
Computer programs were written to calculate and plot the excitation profiles. The sum over u was taken up until u = 6. Inclusion of higher u terms led to no change in the results. The parameters x and y were varied to obtain a good fit to the experimental data. The absorption spectra resulting from a sum of progressions in 2 vibrations was given by the following expression:
Cb)
=
c c u(o(v)2g(u)
Qi,Qz
(5)
u
where g(v) is a line shape function. The line shapes were assumed to be Gaussian with a half-width = y.As the calculated absorption spectra led to poor quantitative agreement with experiment (see text), Lorentzian line shapes were introduced into eq 5. No improvement was observed in the fit.
Results and Discussion and
plus similar terms involving the interchange of i and j and s and e (see ref 10).In this notation, i and j are the vibrational levels of the ground electronic state g, p and u are any of the Cartesian coordinates x , y, or z , R is the dipole moment operator, the u's are vibrational levels of the excited electronic state e with which resonance occurs, Eeu,glis the vibronic energy, Eo is the excitation energy, ha is the (Herzberg-Teller) vibronic coupling operator, Qa is the a t h normal coordinate of the ground state, y is the damping factor related to the half-width of the absorption hand, s is an excited state not in resonance, and finally I go), Ieo), and 1 so) are wave functions for the indicated states. The R term is utilized when resonance enhancement develops because of vibronic coupling between two excited electronic states, while the A term is applicable when only one state is involved in the scattering process. In the present case, the A term was found to give a satisfactory representation of The Journal of Physical Ch8miStry, VOl. 80, NO. 11, 1976
The observed excitation profiles for V I and up a t 23 and -162 "C and for 2vl a t -162 "C are shown as triangles in Figures 2-4. The experimental points for u1 and u:! a t room temperature suggest an intensity maximum near 5200 8, hut no additional resolved structure. In the intensity profiles a t -162 "C, a resolved peak appears around 4600 8, for u1 and 4750 8, for v2 in addition to the main maximum near 5250 8,. For 2u1, the experimental excitation profile has a peak near 4880 8, and shows a second weaker maximum near 5200 8,. The calculated excitation profiles are shown as solid lines in Figures 2-4. For the purposes of calculation, the 0-0 frequency of the electronic transition was set a t 18 850 cm-'. A reasonable fit to the excitation profiles was obtained at both 23 and -162 "C without changing the 0-0 frequency. The excitation profiles of u l (Figures 2a and 2b) were simulated by assuming a value of x = 0.9 for both 23 and -162 "C, with y = 950 cm-' a t 23 "C and y = 500 cm-I a t -162 "C. The calculated excitation profiles show a broad shoulder at room temperature while structure develops a t low temperature. The short wavelength maximum in the excitation profile a t -162 "C arises from the 0-2 transition ( u = 2 in the excited state) while the 0-1 transition appears only as a shoulder. When smaller y values were used, a resolved peak did occur a t the v = 1 level as was found for Y ] of /j-carotene.:' However, the overall fit was better with y = 500 cm-'.
1139
Excitation Profiles of Astaxanthin
tz Lo c W
f
f
-1
w
a
WAVELENGTH, I O
,
l
,
~
l
l
~
J
Figure 4. Observed (A) and calculated (-)excitation
profiles for 2vl at -162 "C.The calculated curve was scaled to equal the observed datum at 4880 A.
>-
t z v)
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W
z
s
WAVELENGTH,
Figure 2. Observed (A) and calculated (-)
profiles for u1 (1524 cm-l) at (a) 23 "C and (b) -162 "C.The calculated curves were each scaled to equal the observed point at 4727 A. I O
I
(
(
(
I
I
I
A
>-
c io
c W
5 w
>
c io
c W
WAVELENGTH,
1
Figure 3. Observed (A) and calculated (-)
excitation profiles for u p (1157 cm-') at (a) 23 "C and (b) -162 "C.Scaled as for v,.
The excitation profiles of u2 were calculated using the same V I at the two temperatures, while the
y values as those for
value of x was 0.8. Again, the calculated excitation profiles shows a shoulder at 23 "C (Figure 3a) but a resolved structure a t low temperature (Figure 3b). The second maximum for u2 a t -162 "C also occurs a t the u = 2 position for this mode. The excitation profile for 2u1 at -162 "C was simulated using identical parameters as those for u1 at this temperature. The qualitative shape (Figure 4) corresponds well with the observation that the intensity maximum at the 0-1 transition is greater than that at the 0-0 transition. The fit is considered satisfactory in view of the experimental error in measuringlow intensities of the overtone. A summary of the parameters used in the calculation for astaxanthin at 23 and -162 "C, as well as those previously reported for @-carotene,is given in Table I. The values of x have been converted to changes in bond length upon excitation by a procedure similar to that described in ref 3. Slight differences are noted for p-carotene and astaxanthin. However, the approximations necessary for the calculation of x in this simple model, namely, that all C=C bonds have the same length and all C-C bonds have the same length and furthermore, that v 1 and u2 are pure C=C and C-C stretching modes, respectively, suggest that these differences may not be significant. The absorption spectrum of astaxanthin a t room temperature, shown in Figure 5a, is devoid of fine structure. P-Carotene (11),on the other hand, has three resolved peaks in the visible r e g i ~ nThe . ~ broad absorption band of axtaxanthin is typical for carotenoid molecules which contain one or more conjugated carbonyl groups.ll At low temperatures, however, fine structure develops (Figure 5b). Absorption in the visible region is known to arise from T-T* transitions of the polyene chain and the fine structure is due to transitions from the ground state to various vibrational energy levels in the first excited state.12 An attempt was made to simulate the observed absorption spectra by assuming that it is composed of a sum of progressions in v 1 and v2. The results are presented in Figures 6a and 6b. The parameters used for each mode were the same as those for the excitation profiles. Qualitatively, the development of structure observed in the absorption spectrum is reproduced by decreasing the half-width from 950 cm-l a t room temperature to 500 cm-l at 162 "C. However, the quantitative agreement is quite poor. There are several possible reasons for this discrepancy: (I) Previous studies on @-carotenehave shown that the v3 vibration contributes in significant fashion to the absorption The Journal of Physical Chemistry, Vol. 80, No. 1 1, 1976
Salares et al.
1140
TABLE I: Parameters Used in the Calculation of Excitation Profiles of YI, v2, and 2 v l of Astaxanthin T , "C
Y,
cm-'
y,cm-'
23 (in CHCI:') -162 (in EPA) -162 (in EPA) 23 (in CHCln) -162 (in EPA)
Astaxanthin 1524 1524 3045 1157 1157
-150 (in isopentane) - 1% (in isopentane)
y-Carotenea 1520 1157
t x,
i
A
E ( +0.023 500
0 r W 4
{-0.025
950
500 250 250
t0.020 -0.031
4000
5000
6000
4000
5 000
6000
Reference 3.
--
I
I
I
I
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W
0
z
a
m
ilI
51
m
a
I
P\ T:23'C
n
I
I
I
WAVELENGTH,
Figure 6. Calculated absorption spectra for astaxanthin. Gaussian line
shapes were used: (a)y = 950 cm-';
W
(b) y
=
500 cm-'.
0
z Q
m
/'
E 0 m
--./
m a
0-0 level in these molecules. The larger value of y for astaxanthin compared to &carotene (500 cm-l in EPA a t -162 O C and 250 cm-' in isopentane a t -150 O C , : ! respectively) is undoubtedly due to the presence of carbonyl groups in astaxanthin. The hydroxyl groups cannot be responsible for the broadening since crustaxanthin (3,4,3',4'-tetrahydroxy-@carotene) shows vibrational structure in i t s room temperature absorption spectrum. The carbonyl groups impart polarity to the molecule as shown by the reduced solubility of astaxanthin in nonpolar solvents. Greater interactions of solvent molecules with astaxanthin in the ground and excited states lead to increased broadening''' of the 0-0 level compared to b-carotene. X-ray crystal studies have shown that the presence of carbonyl groups in the d-ionone rings enhances the noncoplanarity of the rings with the polyene chain.lJ.l5In solutions, the greater noncoplanarity of astaxanthin compared to &carotene may cause a greater distribution of angular conformations about the bond joining the ring and the polyene chain for astaxanthin. This would lead to broadening of the 0-0 transition for the latter molecule. The decrease in width of the 0-0 transition from 950 cm-' a t 23 "C to 500 cm-1 a t -162 "C may be explained by a temperature effect on solvent organization around the solute molecules. In a more rigid environment such as in a glassy medium a t low temperatures, slower solvent rearrangement leads to narrowing of' the 0-0 transition.
' 430c
5002
6500
WAVELENGTH (4)
Figure.5. Absor tion spectra of astaxanthin (a) in CHC13 solution at 23
OC, A 4630%.
4880
8
(b) in EPA solution at -162
OC, , ,A
5240,4900, and
spectrum.:' As the excitation profile for this mode was not measured, no value of x necessary for inclusion in the absorption spectrum calculation was available. A calculation was attempted in which the value of x for vn was taken from the /$carotene work,:' and the calculated absorption spectrum resembled the observed much more closely. (2) Other weak vibrations in the Raman spectrum (Figure 1) were not included in the calculations. T h e extent of their contributions to the absorption spectrum is unknown. (3) The room temperature absorption spectrum is complicated by the presence of molecules which are not in their ground vibrational states prior to excitation. (4) The n--H* transitions of the carbonyl groups may appear in the visible region whereas only the X-T* transition has been considered in the calculated absorption spectrum. The value of y used in the excitation profile calculation provides a measure of the width of the absorption band for the The Journal of Physical Chemistry, Vol. 80, No. 1 1 . 1976
Conclusion It is clear that in circumstances where an unresolved absorption spectrum is composed of more than one vibrational progression and the 0-0 energy cannot be obtained, the res-
1141
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Microwave Spectrum of 1,2,3,6-Tetrahydropyridine
routines, and for considerable advice about the programming.
onance Raman excitation profiles may provide an estimate of this quantity. However, for astaxanthin the spacing between the 0-0,O-1,0-2,.. ., etc. transitions cannot be resolved from the excitation profiles at room temperature. A comparison of the experimental excitation profiles at 23 and -162 "C shows that the development of structure in the resonance Raman excitation profiles parallels its development in the absorption spectrum. It seems apparent for the molecule studied here that Raman excitation profiles depend in a fundamental way on absorption spectral line width and that excitation profiles will not be a general method for resolving vibrational structure of an unresolved absorption spectrum.
(1)(a) NRCC No. 15219.(b) Ph.D. with R. C. Lord, 1972. (2)(a) B. P. Gaber, V. Miskowski, and T. G. Spiro, J. Am. Chem. SOC.,96,6868 (1974);(b) M. Mingardi, W. Siebrand, D. Van Labeke,and M. Jacon, Chem. Phys. Lett.,31, 208 (1975). (3)F. Inagaki, M. Tasurni, and T. Miyazawa. J. Mol. Spectrosc., 50, 286 (1974). (4)F. Galuzzi, M. Garozzo, and F. F. Ricei, J. Raman Spectrosc., 2, 351 (1974). (5)L. Rirnai, R. G. Kilponen, and D. Gill, J. Am. Chem. SOC.,92, 3824 (1970). (6)M. Buchwald and W. P. Jencks, 5iochemistry, 7, 834 (1968). (7)L. Zechrneister, Chem. Rev., 34, 267 (1944). (8)W. Kiefer and H. J. Bernstein, Appl. Spectrosc., 25, 500 (1971). (9) F. A. Miller and B. M. Harney, Appl. Spectrosc., 24, 291 (1970). (10)A. C. Albrecht, J. Chem. Phys., 34, 1476 (1961).
Acknowledgments. We wish to thank Mr. P. Bernath for writing some of the computer programs used in the current work and Dr. W. F. Murphy for the loan of his plotting sub-
210, 139 (1970). (12)J. Dale, Acta. Chem. Scand., 8, 1235 (1954). (13)N. S. Bayliss and E. G. McRae, J. Phys. Chem., 58, 1002 (1954). (14)C. Sterling, Acta Crystaiiogr,,17, 1224 (1964). (15)J. C.J. Bart and C. H. MacGillavry,Acta Crystai/ogr.,Sect. 5, 24, 1587 (1968).
References and Notes
(11) 8.Ke, F. Irnsgard. H. Kjosen, and S. Liaaen-Jensen, Biochim. Biophys. Acta,
Identification and Estimation of the Relative Abundance of Two Conformers of 1,2,3,6-Tetrahydropyridine from the Microwave Spectrum S. Chao, T. K. Avlrah, Robert L. Cook, and Thomas B. Malloy, Jr.*' Departmentof Physics and Departmentof Chemistry, Mississippi State University, Mississippi State, Mississippi 39762 (ReceivedDecember 22, 1975)
The microwave spectra of 1,2,3,6-tetrahydropyridineand its N-d analogue have been studied in the R-band (26.5-40 GHz) and X-band (7-12.4 GHz) regions with a Hew1ett:Packard 8400C Stark-modulated microwave spectrometer. Transitions for two distinct conformers have been assigned and the change in the rotational constants on deuteration has allowed identification of the conformers as half-chair axial and halfchair equatorial. Half-chair refers to the conformation of the ring slfeleton and axial-equatorial to the orientation of the N-H. For the do compound the rotational constants are (in MHz) A = 4897.47 f 0.01, B = 4709.16 f 0.01, C = 2641.48 f 0.01for the axial conformer and A = 4950.50 f 0.01,B = 4743.05 f 0.01, C = 2647.82 f 0.01 for the equatorial conformer. Measurement of the relative intensities of a number of lines yields an estimate of the rather small difference in stability of the two forms of 50 f 30 cm-l (-0.15 kcal/ mol), the equatorial form being the more stable. Because of the distribution of the dipole moment among the principal axis components and the selection rules, the most prominent lines by far are due to the less abundant axial conformer. Under conditions of high resolution several of the lines exhibited resolvable splitting which can be attributed to quadrupole coupling from the nitrogen nucleus. Analysis of the observed hyperfine structure gave xcc = 1.32 f 0.02 MHz; 7xCc= 0.72 f 0.02 MHz for the axial conformer and xcc = -4.48 f 0.04 MHz; vxcc = 0.09 f 0.09 MHz for the equatorial conformer. The drastic difference in the coupling constants for the two conformers is attributed to the difference in the relative orientations of the principal axes of the inertial and quadrupole coupling tensors, respectively.
Introduction .~ As part Of a continuing effort in the study Of the 'Onformations and low-frequency vibrational modes of cyclic molecules, we undertook an investigation of the microwave spectrum of 1,2,3,6-tetrahydropyridine, CsHgN, which is an analogue of cyclohexene. Microwave, far-infrared, and/or Raman studies of cyclohexene, 1,4-dioxene,A2~3-dihydropyran* and A3'4-dihydropyran have been reported'2-s These
studies indicate that the stable conformation is a twisted, or half-chair form. In addition, the barriers to planarity of the rings range from -19 to 25 kcal/mol and the interconversion of the two equivalent stable twisted forms occurs via a bent (half-boat) form with barriers to interconversion ranging from -6 to 10kcal/mol, No direct evidence of the existence of a minimum corresponding to the less stable bent form has been found. It was expected that 1,2,3,6-tetrahydropyridine would also exist with a half-chair ring" skeleton. The Dresence ofthe imino hydrogen, however, renders the two twisted forms nonequivalent, there being two distinct conformers, half-chair axial and half-chair equatorial, respectively. In addition, the The Journal of Physical Chemistry, Vol, 80, No. 11, 1976
