Tarms This is to certify that the thesis entitled RESONANCERAMANS'IUDIES CF CYCLIC AND LINEAR POLYENES presented by DAVID ARI‘HUR GOBELI has been accepted towards fulfillment of the requirements for M.S. Chenistry degree in '” gala! éfvzwf’.. 4-67., / Major professor ”“6 /’ 198%“ i??? L/ /— 0-7639 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation records RESONANCE RAMAN STUDIES OF CYCLIC AND LINEAR POLYENES David Arthur Gobeli A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 1979 ABSTRACT RESONANCE RAMAN STUDIES OF CYCLIC AND LINEAR POLYENES By David Arthur Gobeli Raman excitation profiles were obtained for oxidized heme-a and B-carotene, compounds of both theoretical and biological interest. The tunable excitation radiation was provided by a continuous wave dye laser. The excitation profile of heme-a was taken at selected wavelengths in the visible region of the spectrum in order to facilitate the characterization of two electronic transitions, with absorption peaks at 590 and 5&5 nm, for which the assignments are in dispute. The results suggest that the absorptions correspond to a weak n-n* transition of the cyclic polyene chromophore of the por- phyrin ring and its vibronic sideband. The excitation profile of B-carotene was obtained at closely spaced excitation wavelengths in the preresonance region of the absorption spectrum. Interferences in the David Arthur Gobeli preresonance Raman excitation profile indicate the presence of a low-lying symmetry-forbidden excited electronic state in this linear polyene. TABLE OF CONTENTS Chapter Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . iii LIST OF FIGURES . . . . . . . . . . . . . . . . . . iv CHAPTER I INTRODUCTION . . . . . . . . . . . . . 1 CHAPTER II EXPERIMENTAL . . . . . . . . . . . . . 3 CHAPTER III HEME-A . . . . . . . . . . . . . . . . 12 Theory. . . . . . . . . . . . . . . . . . . . . 15 Experimental. . . . . . . . . . . . . . . . . . 23 ResUlts . . . . . . . . . . . . . . . . . . . . 2“ Suggestions for Further Work. . . . . . . . . . . . . . . . . . . . . . 27 CHAPTER IV B—CAROTENE . . . . . . . . . . . . . . 28 Theory. . . . . . . . . . . . . . . . . . . . . 29 Experimental. . . . . . . . . . . . . . . . . . 32 Results . . . . . . . . . . . . . . . . . . . . 33 Suggestions for Further work 0 o o o o e o o o o o o o o o o o o o o o o 33 APPENDIX - Preparation of Dye Solutions. . . . . . . . . . . . . . . . 36 REFERENCES. . . . . . . . . . . . . . . . . . . . . 37 11 Table LIST OF TABLES Page Laser Dyes Used, Effective Tuning Range and Typical Output Power . . . . . . . . . . . . . . 6 Raman Intensities of OXIdized Heme-a0 e o o o o o o o o o o o 26 iii LIST OF FIGURES Figure Page 1 Dye Laser Configuration. . . . . . . . . 5 I\) Correct Image From the Folding Mirror . . . . . . . . . . . . . . . . . 9 3 Oxidized Heme-a. . . . . . . . . . . . . 13 A Visible Spectrum of Oxidized Heme-a (bis-N-methyl imidazole). . . . . 13 5 Resonance Raman Spectra of Oxidized Heme-a. . . . . . . . . . . . . 25 6 B-Carotene . . . . . . . . . . . . . . . 29 7 Raman Excitation Profile of B-Carotene in Preresonance Region 0 O O O O O O O O O O O O O O O 0 3“ iv CHAPTER 1 INTRODUCTION With the development of the dye laser as a source of continuously tunable monochromatic radiation, the use of resonance Raman spectroscopy as a tool for studying problems of both theoretical and biological importance has grown considerably. The advantages are twofold: the resonance Raman effect selectively enhances the vibra- tions of the electronic chromophore of interest_relative to any others, and because of the corresponding increase in sensitivity, many otherwise inaccessible compounds - notably those of biological significance, which don't occur naturally in high enough concentration to be ob- servable by the normal Raman effect - may be studied.1 In addition, from a plot of the intensity of specific features against the Raman excitation energy information can be gained relative to the electronic transition of interest. This is known as the Raman excitation profile. Moreover, if one studies the Raman excitation profile in the "preresonance" region of the electronic spectrum, an area on the tailing edge of a visible absorption band, electronic states not ordinarily detected by conventional spectroscopic techniques may be observed.2 This form of the excitation profile is useful in detecting low-lying energy states not accessible by electric dipole absorption from the ground state. In this thesis two polyenes, each of both biological and theoretical interest, will be studied. The first, heme-a, is a cyclic polyene found in the majority of living organisms; from its resonance Raman excitation profiles assignments will be made as to the nature of the electronic transitions in the visible region of its spec- trum. For the second compound, B-carotene, the Raman excitation profile in the "preresonance" region of the electronic spectrum will be examined; several vibronic states which are not observed by conventional visible absorption techniques, due to their forbidden nature, will be characterized. CHAPTER 2 EXPERIMENTAL A Raman excitation profile is simply a plot of the scattering intensity of a particular feature plotted against the exciting line wavenumber at constant incident power. Since maintaining constant output power from a dye laser when changing exciting wavelengths and dyes is dif- ficult, a modification of this technique is necessary in order to take this fluctuation into account. This is ac- complished by measuring the scattering intensity of the feature of interest with respect to some internal standard such as a solvent peak, for which the intensity is invariant over the tuning range of interest. The equipment necessary to obtain Raman excitation profiles includes a continuously tunable light source in addition to the optics, monochromator and detection electronics normally required to perform Raman spectroscopy. Laser light whose output wavelength can be varied continu— ously over a wide range can be attained from a continuous wave (cw) dye laser. From a tunable dye laser, relatively high-power radiation can be attained for use as a mono- chromatic Raman excitation source in wavelength regions inaccessible by conventional fixed-frequency high-powered gas lasers, such as the argon or krypton ion visible lasers. Although the cw dye laser in this laboratory is similar to commercially available models, it pOSSesses several unique features, so its operation will be detailed.3 High intensity pumping radiation, typically the all- lines output from either an argon or krypton ion laser, is necessary in order to produce lasing within a dye solution. The pump used in this experiment is a Spectra- Physics Model 16“ argon ion laser producing typically A-S watts of output power when used in the "all-lines" mode. This output is focussed onto a flowing Jet stream of laser dye which is continuously circulated in order to prevent the rapid heating, and subsequent rapid degrada- tion of the dye solution, which would otherwise be caused by utilizing a stationary dye cell in the focus of the pumping laser. In addition, the dye solution is cooled, usually with dry ice. A basic three-mirror cavity is constructed around the dye Jet as shown in Figure 1.3 The cavity consists of front, back and folding mirrors obtained from commercial sources. This configuration is used in preference to a standard two-mirror cavity in order to compensate for the astigmatism caused by having the dye jet at Brewster's angle with respect to the FOLDING MIRROR — — - ----- - - A PUMP LIGHT N - - _ ______ ’ ’ 2:532?” OUTPUT , ’ [:3] MIRROR /,’oye* FOCUSSING \(t LENS BACK MIRROR *Flow of dye solution is perpendicular to page Figure l. Dye Laser Configuration. incident pumping radiation, and to eliminate the need of using a focussing mirror instead of the focussing lens which is employed in the three-mirror cavity. A diffrac- tion grating which is placed after the output mirror is used to tune the output of the dye laser and provides a typical bandwidth of 0.1 nm. A Spectra-Physics Model 376 dye circulator and nozzle are used to produce the dye jet stream. In order to maintain a uniform dye film through the active region of the cavity it is necessary to use a viscous solvent for the dye. For the dyes used in this work the solvent chosen was ethylene glycol or an ethylene glycol/benzyl alcohol mixture. Table 1 lists the dyes used and the wavelength region over which each could be effectively tuned. With the exception of Table 1. Laser Dyes Used, Effective Tuning Ranges and Typical Output Power. Typical Output Dye Tuning Range Power Coumarin—6 522-552 nm 60—120 mw Disodium fluorescein SAC-575 nm l20-AOO mw Rhodamine-6G 570-6u0 nm 300-600 mw Rhodamine-6G, it is necessary to add varying amounts of cyclooctatetraene (COT) to the dye solution; COT serves as a triplet quencher for the dye and thus enables lasing. The exact preparation method for each dye solution is outlined in the Appendix. The tuned laser radiation is collected off of the grating and is used directly by way of completely reflect— ing mirrors which serve as beam directors. This method is not quite as convenient as that outlined in R. J. Thrash's thesis,3 where a beamsplitter was employed within the cavity to eliminate the need for adjusting the beam directing optics as the excitation wavelength is changed. However, it is superior in that the usable output power is much higher. The power available with this-configura- tion ranged typically from 100-800 mw gs. S-AO mw attained by Thrash. This increase of more than an order of magni- tude of usable output provides a substantial increase in the signal-to-noise ratio in the Raman spectra Obtained. The output power from all dyes remained stable for many hours. Since the cw dye laser used in these experiments is "home-made" and easily disassembled, the cavity align- ment procedure will be outlined in detail. The optical mounting equipment used in this dye laser is designed for easy interchange of mirrors. This is necessary because each specific tuning region requires specifically coated optical substrates which optimize the output power for the particular dye being used. The back mirrors used (Coherent Radiation) have a focal length of 50 mm and the folding mirrors have a focal length of 75 mm. All are 0.5 inch in diameter and are coated to provide maximum reflectivity at the lasing wavelengths of the particular dye being circulated. The output mirror, sometimes re- ferred to as the transmitter, or output coupler, is flat and is usually 95—98% reflecting, depending on the mirror being used and the wavelength at which the laser is operating. The focussing lens used for the argon ion beam is an achromat with a focal length of 22 mm and a diameter of 10 mm. A step-by-step procedure for the align- ment procedure follows. Step 1. With the argon ion laser operating at minimal power, position the output so that it strikes the flowing dye Jet about 0.5 cm or so from the nozzle at an approxi- mate right angle. All other optical elements should be removed at this step. Step 2. Rotate the nozzle of the dye circulator so that the right angle between the argon ion beam and the direction of the flow is maintained, but the reflected laser intensity is minimized. This should be attained at Brewster's angle. Step 3. Insert the achromatic lens and bring the argon ion beam into sharp focus onto the dye Jet. The reflection, which will be observed on the ceiling, should now look like two round spots which may or may not be coincident. If the argon ion beam is imprOperly focuSsed, the spots may not be round. Step 4. Insert the output mirror about 1 foot from the dye Jet and position it vertically so that its center is approximately coplanar with the dye Jet and argon ion beam. Step 5. Now insert the folding mirror in the dye cavity so that it is as close to the plane defined by the dye Jet intersecting the argon ion beam as possible. Its approximate position will be directly above the focussing lens. Now adJust the folding mirror so that the light from the dye Jet is reflected onto the output mirror. AdJusting the distance of the folding mirror from the dye Jet, either vertically or horizontally, will change the shape of the spot hitting the output mirror. Now adJust the output mirror so that the spot is reflected onto a card mounted on top of the argon ion laser. Then position the folding mirror so that the shape of the image is ap- proximately the same as is shown in Figure 2. 7 Figure 2. Correct Image From the Folding Mirror. Step 6. This is the most difficult step. The back mirror is now inserted so that its center is approxi- mately colinear with the folding mirror and the active region of the dye Jet. The light reflected from the back mirror must now be passed back through the active region of the dye Jet (where the argon ion light is focussed) onto the folding mirror, then onto the output mirror, and then onto the card. Once this is done, adJust its position so that the image caused by this mirror is the same shape as the one from the folding mirror. Then superimpose 10 the two images. Step 7. Now tilt the output mirror so that the two superimposed spots fall on the folding mirror. The dye laser should now lase. At this point however, the radia- tion is not tuned. Step 8. To achieve tunability, the grating should be inserted close to the output mirror so that the first- order reflection of the dye laser output re-enters the system. Now a slight rotation of the vertical axis of the grating should tune the dye laser output. Once the radiation is tuned, it can be directed into the sample compartment of the monochromator. The direc- tion of the polarization of the dye laser light will be the same as the argon ion laser light. The light is focussed by way of bottom illumination so as to be parallel to the entrance slit of the monochromator. The scattered radiation is collected, passed through a polarization scrambler, and finally onto the entrance slit of a Spex- Ramalog double monochromator. The scattered radiation is detected using an RCA C3103UA photomultiplier tube cooled by a Products for Research TE-lODTS thermoelectric cooler, typically maintained at -20 degrees C to minimize the dark current. The phototube housing circuitry is de- signed to Operate in either a do or photon counting mode. If the Raman scattering is sufficiently strong, the system is kept in the dc mode, with between 1900 and 2000 volts 11 applied phototube voltage and 3.0 x 10.8 amp full scale output presentation on a chart recorder. For weakly scattering samples photon counting, usually with similar ’4 voltages and 3.0 x 10 cps full scale deflection, is employed. CHAPTER 3 HEME-A The heme—based proteins are a class of compounds of tremendous biological interest, of which hemoglobin and A They consist of cytochrome are two important examples. an iron-porphyrin moiety either chemically bonded or electrostatically attached to a larger protein super- structure. It is the heme portion of the protein which gives rise to the absorption maxima in the visible region of the spectrum. Hence, by use of the resonance Raman effect, it is possible to gain information about the porphyrin portion of the protein, or as described in this thesis, the isolated metalloporphyrin. Many organisms contain cytochrome oxidase, which is necessary to mitochondrial respiration. Heme-a is the protein bound porphyrin which gives rise to peaks in the visible spectrum of cytochrome oxidase. By isolating this compound and applying resonance Raman spectroscopy to it, well-founded assignments can be made of the particular electronic transition giving rise to each of the visible absorption peaks. Oxidized heme-a, whose structure is shown in Figure 3 l2 l3 fo CH0” CH3 Hf CH I CH2 OHC C”3 fiHz |H2 9*2 9'2 COOH COOH Home O Rs. -CHzCH-(':CH2CH2CH8(|ZCH2CH2CH'(':CH3 CH3 CH3 CH3 Figure 3. Oxidized Heme-a. OXIDIZED HEME-A 2! 9 p. O. Q: C) ‘é I 8 T O o l l l .1 ll 400 450 500 550 600 650 700 x(nm) Figure N. Visible Spectrum of Oxidized Heme-a (bis—N- methyl imidazole). 1& has a visible absorption spectrum consisting of three main features, as shown in Figure &. The most prominent absorption, an intense band center- ed at &22 nm with an extinction coefficient of about 105 is widely accepted as a strongly allowed n-w* transition in the l6—membered porphyrin ring. This feature is com- monly referred to as either the B(0,0) or Soret band.5 Two much weaker features occur between 500 and 600 nm. Each has an extinction coefficient approximately one order of magnitude less than that of the Soret band. The assign- ment of the electronic transition corresponding to each of these bands is a subJect of debate at the present time. One theory assigns the low energy band at 590 nm, based upon magnetic circular dichroism (MCD) studies, to a charge-transfer band between the porphyrin ring and a formyl substituent attached to it.6 The remaining feature at 5&5 nm is assigned to a forbidden n-w* transition. The other prominent theory attributes the low energy feature at 590 nm to a symmetry-forbidden n-n* transition of the porphyrin ring, the remaining feature at 5&5 nm being assigned as a vibronic sideband of the feature at 590 nm. In this model the 590 nm is assigned as the a or Q(0,0) band, and the 5&5 nm feature is termed the B or Q(1,0) band.7 ' From Raman spectra obtained with excitation wavelengths corresponding to approximately 590 and 5&5 nm it can be 15 readily determined, by observing which vibrational features are enhanced, which assignment is consistent with reson- ance Raman scattering theory. Theory The connection between the excited state electronic wavefunction and the observed enhanced intensities in various ground state vibrational modes arises from the vibronic coupling terms in equations describing the resonance Raman effect. A brief discussion of the non- resonance Raman effect is given first and then modifica— tions are made for resonance Raman scattering.8 For the transition g + f, the Raman scattering inten- sity per molecule averaged over all angles is given by: + n(w-w ) — — :8 f = K IO ZIIcazmpg’fl2 (1) pa IO represents the intensity of the incident radiation, ka = E-Eg, a is known as the scattering tensor and no is related by a multiplicative factor and the vector po- tential to the polarizability, and w is the (angular) frequency of the incident radiation. a is given by the po Kramers-Heisenberg dispersion formula: l6 _ E 00 - “ i ET-Eg-Mw + (2) 1 E -Eg+Mw The index I represents a summation over all intermediate states. It should be noted that far from resonance I’ E -Eg-hw >> 0 and both terms in the equation will con- tribute to the intensity of scattered radiation. When resonance is approached ETIEE-Mw = 0 and a modi- fication must be made to prevent the denominator in the first term of the scattering tensor from becoming zero. This is accomplished by adding a damping factor if;, which takes into account the finite lifetime Of the ex- cited state |I>. The scattering tensor now becomes: g’f - (Goo) ’ + _ _ <3) Ei-Eg+Mw-1P1 Assuming that the damping factor is small, when the system is near resonance the first term will completely dominate. 17 Thus under the condition of resonance: )R,? = (El plT> (14) fi I 22. 00' _ I E -Eg-Mw-iI'-f The terms |g>, |I>, and If> represent the total wavefunc- tion of the system. Under most circumstances the SchrO- dinger equation cannot be solved in this form, and approxi- mations must be introduced which separate the wavefunction into a product of vibrational and electronic terms. Utilizing the Born—Oppenheimer approximation one can write the complete wavefunction as a product: |g> = |g>|n> (5) The term |g>|n> represents a product Of a purely electronic term |g> with a purely vibrational term |n>. Thus, each matrix element within the polarizability tensor can now be written as the product of strictly vibrational and electronic integrals; it is assumed that fip operates only on the electronic coordinates. The polarizability tensor may now be represented as a product of electronic and vibrational wavefunctions: 18 iv_ gn_ _I'I E E Kw 11v gn;fn' (o. a) = 22 9 iv 0 p I = 22 MgiM1f 6 iv ( ) iv gn E -E -Mw-if1v For the normal resonance Raman effect |g>, the initial electronic state, will be the same as the final electronic state If>. Thus: 0 p ( )gnggn' = 22 MgiMiR (7) ape iv iv_ gn_ _ E E Kw iI‘iV The symbols M21 and Mgg represent electronic transition dipoles and will be equal if both |g> and Ii> represent O E parametrically dependent upon Q, the vibrational co- real wavefunctions. The matrix elements M 1 and Mgg are ordinates, and are expected to vary weakly with small changes in the equilibrium nuclear coordinates. In order to account for these changes and any consequences which they might bring, it is necessary to expand the matrix elements MG and MD in a Taylor series around the equi- librium nuclear coordinates which will be represented 19 by Q = 0: C MO(Q£) = Mg(Qg=0) +(93LJ BQE Q5 = O 2 2 o Q + (a M ) —%— + . . . (8) 3Q 2 Qg - 0 Truncating the series after the first term and using the shorthand notation: E 0 (g%—) = 0(1) € Q€=0 one has: 0(Qg) = 0(0) + 0(1)Qg (9) Analogously: 0(Qg) = 0(0) + 0(1)Qg It should be noted here that QE implies summation over all normal modes of vibration. The scattering tensor can now be expressed as a sum of four terms: 2O 1 gn gn' _ iv gn_ _ (ape) ’ - I g g E -E Kw iI‘iv x [0(O)p(0) + 0(l)p(o)§n|Q€|V> + 0(O)p(1) + p(l)c(l)] (10) (1) o and p (1) can be obtained from perturbation theory: <1IuoIs> g o I (11) =1 Ei-ES 0(1) =2 E S (1)_ A similar expression is obtained for p Neglecting the p(1)0(l) term in the scattering tensor, the summation can be divided up into two parts. The first part will contain the p(0)o(0) term and will not vibronically couple any excited electronic states: Apd 'MD M0 = Z I gi 1% (12) i v Ei-Eg‘Mw-ir 1v 21 This term is commonly referred to as Albrecht's A term.9 Note that the A term contains the product of electronic transition moments and vibrational overlap integrals. For this reason this part is sometimes called the Franck- Condon term. The second and third terms which contain p(1)0(0) and p(O)o(l) combine to yield Albrecht's B term: 2 z :2 ”Vi? DO i sii v E (El-Es)(EiV-Egn-Mm-ir III II iv) >< [MZngg + MD M0 g1 sg40m v * 65% I sag H a 0 LOCI- Aigsuaiul UDUJDH 26 Table 2. Raman Intensities of Oxidized Heme—A. Aex=583 nm Aex=5&2 nm (cm-l) AU(cm'l) 16&0 w l6&0 s 1616 w 1587 vs 1586 vs 155& s 155& m 1515 s 1512 w 1&71 w 1369 w 137& w 1336 w 1328 w 1310 S 1311 m 1286 vw 1289 m vs (very strong) vw (very weak) 5 (strong) m (medium) w (weak) 27 heme-a vibrations in the ground state lie in this wave- number range, and their frequencies would be expected to change very little in a low—lying w* excited state. Suggestions for Further Work It is suggested that a more accurate characteriza- tion of heme-a would result if more excitation wavelengths, intermediate between 590 and 5&5 nm were used to obtain resonance Raman spectra. Then definite trends in peak intensity might be observed as excitation frequency is incrementally changed, and further conclusions may be drawn. This can be readily accomplished with the experi- mental apparatus in its current form. A complete polarization study would yield additional information as to which resonantly enhanced features in the Raman spectrum under visible excitation arise from Albrecht A-terms and which come from B-terms. This would then facilitate the assignment of the various features shown in Figure 5 to specific modes of vibration. CHAPTER & B-CAROTENE The class of compounds known as the linear polyenes, to which B-carotene belongs, is characterized by the pres- ence of a chain of alternating single and double carbon- carbon bonds. Various theoretical works have hypothesized the presence of a low-lying, symmetry-forbidden transition to the red of the first allowed absorption peak, which occurs at about &83 nm in B-carotene.10 It has been sug— gested that the presence of such a low-lying electronic state may play an active role in the energy transfer from carotenoids to chlorophyll in photosynthesis.11 Hence, it is important to locate and characterize this "phantom state". B—carotene, whose structure is shown in Figure 6 is non—fluorescent and thus two-photon fluorescence (TPF) methods cannot be applied to help locate this state. Because the concentrations necessary to make a two-photon absorption measurement of this state cannot be attained, indirect methods of observation must be sought. One such method is the preresonance Raman excitation profile. Through vibronic coupling, features are seen superimposed on the background of the preresonance portion of the 28 29 [III] ‘\. ~\ ‘\ ‘\ \\ ‘\ \\ \g '\t [II' VIRTUAL CENTER OF SYMMETRY Figure 6. B-Carotene. excitation profile which may be attributed to these hidden states. Theory The theoretical basis for the modulation of the normal preresonance enhancement, due to a symmetry-forbidden transition can be demonstrated simply by expanding the modified scattering tensor used for resonance Raman scatter- ing as shown in Friedman and Hochstrasser.l2 A simplifica- tion is made by assuming that the summation contains only two terms. One indicates the low-lying "phantom" state and the other indicates the dipole—allowed state: = Z 8° i Ei-Eg-Mw-ir1 “1 + a2 (1&) AEl-ifl AE2-1F2 30 The Raman scattering intensity due to these states is then proportional to the square of the scattering tensor: I W Z|(<:Ic>c)|2 (15) OO Here a1 and a2 are the geometric mean of the dipole strengths for the transitions connecting the initial and final states via intermediate states one and two, which correspond to the forbidden and allowed electronic states, respectively. Expanding this term leads to the following equation: oi GS 2d1a2 I” 22" 2-—2+ (15) AE1+P1 AE2+f2 AEIAE2+P1F2 It has been assumed that all states are real. All im- aginary cross terms are neglected. When the excitation energy is close to the energy separation between the "hidden" and ground states, the first term is the reson- ance term, which is insignificant due to the forbidden nature of al. The second term is a preresonance term due to the nearby allowed electronic transition. The third term, sometimes referred to as the "interference" term, is due to both the forbidden and allowed electronic states and is of significant magnitude due to the cross 31 product of o1 and a2. Neglecting the resonance term as insignificant, the sum reduces to Just two terms a2 2d a I I —-§——— + 1 2 (17) The first term will produce a steadily rising background as the exciting energy approaches the allowed electronic transition. The second term should produce a fluctuation superimposed upon this steadily rising background as the excitation energy becomes coincident with the forbidden transition. This feature would be an indication of a hidden vibronic state. The experiments of R. J. Thrash,3 which were performed under a similar set of experimental conditions but using a different lasing medium, revealed several features in the preresonance Raman excitation profile in the region between 18200 cm"1 and 19000 cm'l. These features were hypothesized to represent interference patterns caused by "hidden" vibronic states.2 The previous study of B-carotene was performed using state-of-the-art instrumentation. Several different dyes were employed, each pushed to the limit of its tuning range. (For example, Thrash obtained barely usable laser power from disodium fluorescein in a region more 32 the 100 A to the blue of the limit prescribed by most com- mercial suppliers.) The Raman spectra were obtained with a single monochromator, which has inferior stray-light reJection compared to the double monochromators normally used. The purpose of the current repetition of that in- vestigation, with an improved laser, and employing a single (different) dye with a broad tuning range as well as a double monochromator, was to assure that instrumental artifacts did not contribute to the previous results. Experimental Synthetic B-carotene was Obtained from commercial sources (Eastman) and used without further purification. Spectral grade cyclohexane was used also without further purification. The concentration was determined to be 1 x 10-” M from the Beer's law plot in R. J. Thrash's thesis.3 Coumarin-6, which served as the lasing medium for this experiment, provided a tuning range of 522-552 nm; typical output power ranged from 80-120 mw with a spectral bandwidth of 0.1 nm. The raman scattering from the sample at Av = 1525 cm-1 (the C=C symmetric stretch) was strong and hence a lower phototube voltage (~1600 V) could be used than in the previous experiment; and resulted in an improved signal- to-noise ratio. Monochromator slits were set at 33 300/600/300 (um). The electrometer was used at 3 x 10"8 amp full scale deflection. At each excitation wavelength the ratio of the peak area of the B-carotene band at 1525 cm"1 to that of an 1 was determined. internal standard solvent band at 1&&5 cm- Measurement of peak areas was made by triangulation. Typical reproducibility was i5—10%. Results The Raman excitation profile thus obtained for B-caro- tene is shown in Figure 7. Asterisks indicate features reported by Thrash, 22 al., for comparison the previous excitation profile is reproduced in the inset. Since this part of the thesis is essentially a replication of Thrash's previous work, a detailed inter- pretation will not be given. The results found in this study closely resemble and hence tend to confirm those of Thrash; the preresonance Raman excitation profile shows interference effects which are likely due to vibronic states of a "hidden", symmetry-forbidden excited electronic state having its origin near 17,000 cm.-l Suggestions for Further Work It has been demonstrated that the exact location of the low-lying forbidden state in linear polyenes is a .QOHwom mocmcomopmam a“ ocouonmoIm no mafimonm COHumwfioxm cmsmm .u opswfim 9.83 xop 000.0. 000.0. 000. Q. _ _ _ _ . _ _ _ _ _ A .163 Ron I... D.- a 000. o. 03.9 08.0. m 14 _ d M w .. . I on m. m. . a w . . . Ind m. m . . . t» u m. o 0.. o o. e 0 III. 0.” m. w . t» u A... m * 6 I In» w. I? . . a . * GM. 106 W Ia . . .0. a a I 0.0 n t» m. linvn 35 function of the number of double bonds in the polyene.13 Quantum mechanical calculations indicate that as the number of double bonds is increased, the low-lying state should drop in energy until it asymptotically reaches a limiting value.10 This limiting value is predicted to be reached at approximately eleven double bonds. Depending on whether the double bonds in the terminal rings are counted or not, B-carotene has 9 or 11 conJugated double bonds. For comparative purposes, it would be interesting to obtain the preresonance Raman excitation profile of lycopene, a commercially available carotenoid with 11 conjugated double bonds and no terminal rings. APPENDIX PREPARATION OF DYE SOLUTIONS Coumarin 6 - For each liter of final solution, 0.&& grams of the dye should be dissolved in 150 ml of methanol mixed with 150 m1 of benzyl alcohol. After the dye is dissolved, which might take 2& hours, 700 ml of ethylene glycol and 3 m1 of cyclooctatetraene are added. The entire solution should be filtered by Buchner funnel before being added to the circulator. Disodium fluorescein - For each liter of final solution. 1.1 gm of the dye should be dissolved in 50 ml of methanol and added directly to 950 m1 of ethylene glycol. &-5 Drops of COT should be added per liter. Rhodamine-6G - For each liter of solution 0.96 gm of Ithe dye should be dissolved in 50 ml of methanol and added directly to 950 m1 of ethylene glycol. Several drops of COT may be added per liter to increase the output power, but this is not necessary and will decrease the lifetime of the dye. 36 REFERENCES 2. 7. REFERENCES R. J. Clark, and B. Stewart, Structure and Bonding, 39. 3 (1979). R. J. Thrash, H. L.~B. Fang and G. E. Leroi, J. Chem. Phys. £1, 5930 (1977). R. J. Thrash, Ph.D. Thesis, Michigan State University (1977). T. G. Spiro and T. M. 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