Excited-State Dynamics of Carotenoids Studied by Femtosecond Transient Absorption Spectroscopy

Abstract

Carotenoids, natural antenna pigments in photosynthesis share a symmetric backbone of conjugated polyenes. Contrary to the symmetric and almost planar geometries of carotenoids, excited state structure and dynamics of carotenoids are exceedingly complex. In this paper, recent infrared and visible transient absorption measurements and excitation dependent dynamics of 8'-apo-${\beta}$-caroten-8'-al and 7',7'-dicyano-7'-apo-${\beta}$-carotene will be reviewed. The recent visible transient absorption measurements of 8'-apo-${\beta}$-caroten-8'-al in polar and nonpolar solvents will also be introduced to emphasize the complex excited-state dynamics and unsolved problems in the $S_2$ and $S_1$ excited states.

Introduction

Carotenoids play important roles in photosynthesis; they collect the blue-green region of sun-light in which chlorophylls have no absorption, and participate in the photoprotection in excess-light condition by quenching the singlet excited states of chlorophylls.1-5

The electronic structure of carotenoids are based on a C2h symmetry of all-trans-polyene backbone and well represented by the simple energy level scheme of the singlet excited states S2()and S1 () and the ground state S0)()1,6 The S2 state populated by one-photon excitation rapidly relaxes to the S1 state via a putative conical intersection 7,8 and the energy levels and dynamics of these singlet excited states have frequently been interpreted by the energygap law.9 Another dark state () lying between the S2 and S1 states have been reported in numerous studies and often denoted as or S* although they are not yet confirmed as separate electronic states.10-12

Carotenoids with electron-withdrawing groups such as carbonyl or cyano group show strong charge transfer character in the S1 excited states in polar solvents, which lowers the energy level of the S1 states and facilitates the internal conversion to the ground electronic states.13,14

The excited-state dynamics of 8'-apo-β-caroten-8'-al and 7',7'-dicyano-7'-apo-β-carotene have been investigated by applying various pumping methods in time-resolved vibrational and infrared transient absorption measurements.13,15-19 One-photon excitation at the lowest vibrational level of the S2 state would populate the bottom of the S2 state and twophoton excitation at the half of the level of the S1 state, where the carotenoid has no one-photon absorption would populate the S1 state directly. When one-photon excitation aimed to a higher vibrational level of the S2 state, the S2 state with excess vibrational energy can be created.11,17 A model of two non-communicating minima on the S1 potential energy surface would explain the distinct relaxation lifetimes and pathways following one-and two-photon excitations. The second minimum of the S1 state which generates a charged radical species in the ground state might be understood as one of conformational isomers in the singlet excited state of the conjugated polyene backbone.

In this paper, we review recent progresses on the excited state dynamics of polar carotenoids, 8'-apo-β-caroten-8'-al and 7',7'-dicyano-7'-apo-β-carotene and also show the recent transient absorption measurements on 8'-apo-β-caroten-8'-al in the visible wavelengths.17-19

 

Experimental

A Ti:sapphire regenerative amplifier (Libra-HE-USP, Coherent) generates pulses with 805 nm center wavelength, 50 fs pulse widths, and 3.5 mJ pulse energy at 1 kHz repetition rate. A home-built noncollinear optical parametric amplifier (NOPA) generates pump pulses with a variable wavelength of 450-650 nm.20 NOPA requires seed and pump pulses. Seed pulses are generated by the supercontinuum generation in a sapphire window and pump pulses by the second harmonic generation in a β-barium borate (BBO) crystal (θ = 29.2°, φ = 90°, 0.1 mm thick, Eksma). The fundamental pulse energy was controlled by a half wave plate mounted on a rotational mount and a cube polarizer. In this homebuilt NOPA, the 403 nm pump pulses are divided into signal and idler pulses in visible and near-infrared wavelengths, respectively, in the second BBO crystal (θ = 26°, f = 90°, 2 mm thick, Eksma). NOPA output was optimized by adjusting the overlap angle between seed and pump pulses, the angle θ of BBO crystal, and the time delay between the seed and pump. The output from NOPA was compressed to remove extra chirps by a prism-pair compressor. The pulse widths of NOPA output pulses were measured with a homebuilt interferometric autocorrelator based on two-photon absorption in an UV photodiode (SiC, Electro Optical Components) and pulse widths less than 35 fs were obtained throughout the NOPA output wavelengths.21

Supercontinuum whitelight probe pulses were generated by focusing the fundamental pulses (∼1 μJ) into a sapphire window (3 mm thick, Eksma) by using a R = 10 cm dielectric concave mirror. The intensity and beam size of the fundamental pulses were controlled with a spatial filter (iris) and a neutral density filter to generate stable white light pulses and the white light was re-collimated with another R = 10 cm concave mirror with a protected silver coating. A supercontinuum probe pulses covers a broad wavelength range of 450-900 nm.

Acton SP-150 spectrograph ( f = 150 mm) from Princeton instrument with a 300 gr/mm grating was used for transient absorption measurements. A photodiode array with two rows of Hamamatsu S3094-1024Q purchased from Spectronic devices Ltd. offers the shot-to-shot measurements with 1 kHz repetition rate. Each spectrum taken by the photodiode array is modulated with an optical chopper (MC-2000, Thorlabs) running at 500 Hz (half of the repetition rate of the pump pulses). To evaluate the change in absorbance (ΔA) of the sample by the pump pulses, probe spectra measured in even and odd sequences are averaged separately then ΔA can be calculated by the following equation.

A retroreflector on a computer-controlled translational stage changes the optical delay between the pump and probe pulses. The further details of the transient absorption setup are explained elsewhere.22

8'-Apo-β-caroten-8'-al and solvents are purchased from Santa Cruz Biotech and Sigma-Aldrich and used without further purification. Sample concentrations of 2.5∼5 × 10−5 M were used with circulation and the excitation pulse energies of < 200 nJ were used for transient absorption measurements.

The laser setup and details of the infrared transient absorption spectroscopy have been described in detail elsewhere.19

 

Results and Discussion

Steady-state Absorption. Figure 1 shows the steady-state absorption spectra of 8'-apo-β-caroten-8'-al and 7',7'-dicyano- 7'-apo-β-carotene in cyclohexane and chloroform solutions. The S0 → S2 absorption band of 7',7'-dicyano-7'-apo-β- carotene in chloroform (λmax = 560 nm, 170 nm FWHM) is broader and red-shifted from that of 8'-apo-β-caroten-8'-al in chloroform (λmax = 475 nm, 100 nm FWHM). The absorption spectrum of 8'-apo-β-caroten-8'-al in cyclohexane clearly shows three resolved vibronic bands at 487, 460, and 435 nm, which are assigned as the 0-0, 0-1, and 0-2 vibronic bands of the S0 → S2 transition. The absorption spectrum of 8'-apo-β-caroten-8'-al in cyclohexane is slightly blue-shifted from the spectrum in chloroform, which is arising from the difference in the solvent polarizability.17

Figure 1.Steady-state absorption spectra of 8'-apo-β-caroten-8'-al in cyclohexane (solid) and chloroform (dashed) and 7',7'-dicyano- 7'-apo-β-carotene in chloroform (dotted).

The S2 states of 8'-apo-β-caroten-8'-al and 7',7'-dicyano- 7'-apo-β-carotene were reached by one-photon excitation (1PE) at 490 and 508 nm, respectively, and the S1 states of both carotenoids were reached directly by two-photon excitation (2PE) at 1275 and 1400 nm, respectively. It is clear that there is no absorption around 638 nm (half of 1275 nm) for 8'-apo-β-caroten-8'-al, whereas 7',7'-dicyano-7'-apo-β- carotene has some absorption at 700 nm (half of 1400 nm). However, we expect 2PE at 1400 nm to only excite the S1 state of 7',7'-dicyano-7'-apo-β-carotene based on the selection rule.

Infrared Transient Absorption Measurements: 1PE vs. 2PE. Figures 2(a) and 3(a) show infrared transient absorption spectra of both carotenoids in chloroform solutions measured with mid-infrared probe pulses in the frequency range of 1350-1800 cm−1 following the S2 excitation. The S2 state evolves into the lower S1 state via conical intersection 7,8 in ultrafast lifetimes (∼100 fs for 8'-apo-β-caroten-8'- al23), so the infrared spectra shown in Figures 2(a) and 3(a) with 1PE represent those of carotenoids in the S1 state. The details of band assignments can be found elsewhere,19 but it can be summarized with ν(C=O) at 1683 cm−1 and three ν(C=C) at 1488, 1550, and 1585 cm−1 for 8'-apo-β-caroten- 8'-al. All of these bands decay with 19.4 ps lifetime,19 which was confirmed as the S1 lifetime by visible transient absorption measurements.13,15,24,25 The ν(C=C) bands of 7',7'-dicyano- 7'-apo-β-carotene in the range of 1450-1700 cm−1 decays with a 2.0 ps lifetime of the S1 state.26

Transient infrared absorption spectra of two carotenoids following the 2PE at 1275 and 1400 nm are shown in Figures 2(b) and 3(b). Transient infrared spectra at early time delays (< 20 ps in Figure 2 and < 3 ps in Figure 3) are very similar between 1PE and 2PE. The S1 infrared bands of 8'-apo-β-caroten-8'-al in Figure 2(b) show a similar 17 ps lifetime, which is slightly shorter than the results with 1PE at 490 nm. The S1 bands of 7',7'-dicyano-7'-apo-β-carotene in Figure 3(b) show a 1.7 ps lifetime, also slightly shorter than that of 1PE results.

Figure 2.Infrared transient absorption spectra of 8'-apo-β-caroten- 8'-al in chloroform with (a) 1PE at 490 nm and (b) 2PE at 1275 nm (reproduced from refs. 18,19).

However, transient absorption spectra of 8'-apo-β-caroten- 8'-al and 7',7'-dicyano-7'-apo-β-carotene at longer delays (after ca. 50 and 5 ps, respectively) with 2PE are clearly different from the spectra observed with 1PE. A very strong transition around 1510 cm−1 appears for both carotenoids with multiple time constants (∼15 and ∼140 ps for 8'-apo-β- caroten-8'-al and ∼30 and ∼200 ps for 7',7'-dicyano-7'-apo-β- carotene19) and does not show a decay in 1 ns time delay in both carotenoids.

Infrared transient absorption measurements of both polar apocarotenoids following 1PE on to the S2 state and 2PE on to the S1 state clearly show that there are more than two minima in the S1 states of these carotenoids and that the second minimum is not populated during the internal conversion from the S2 state via the conical intersection. The second minimum of the S1 states of both carotenoids show a distinct relaxation pathway from that of the S1 minimum, which forms a long-lived species in the ground electronic state S0.

Infrared Transient Absorption Measurements: Excess Energy 1PE. Intermediate dark states between the S1 and S2 states of carotenoids are often found when the excited states of molecules are populated with excess vibrational energy. Polivka and co-workers showed that the excited state absorption from the intermediate S* state of zeaxanthin increased with increasing excess vibrational energy above the bottom of the S2 state.11 Larsen and co-workers found another state (claimed as the state) which showed a longer decay time than that of the S1 state in β-carotene with 400 nm excess energy excitation.10

Figure 3.Infrared transient absorption spectra of 7',7'-dicyano-7'- apo-β-carotene in chloroform with (a) 1PE at 508 nm and (b) 2PE at 1400 nm (reproduced from refs. 18, 19).

Transient infrared absorption measurements with an excess energy excitation at 405 nm on to the S2 state of 8'-apo- β-caroten-8'-al17 are shown in Figure 4. As shown in the steady-state absorption spectrum, the excitation at 405 nm might excite the 0-3 band of the S2 state of 8'-apo-β-caroten- 8'-al in chloroform. One ν(C=O) and three ν(C=C) bands well represent the infrared spectrum in the S1 state (shown in Figure 2(a) with 490 nm excitation). However, transient absorption signal existed even long after the S1 decay especially around 1510 cm−1 and the infrared absorption spectrum observed with 405 nm excitation after the S1 decay was almost identical to that measured with 2PE at 1275 nm (Figure 2(b)). The kinetics of the 1512 cm−1 band in Figure 4 shows 18.4 ps decay (close to the S1 decay lifetime) but about 13% of population shows no decay in 1 ns (infinite lifetime). These infrared spectra are compared in Figure 5 along with the simulated infrared spectrum of 8'-apo-β- caroten-8'-al by the density functional theory (DFT) method.18 Details of the DFT simulation and the nature of the longlived species observed with 2PE and 1PE with excess vibrational energy will be discussed later in a separate section.

Figure 4.Infrared transient absorption spectra of 8'-apo-β-caroten- 8'-al in chloroform with 1PE at 405 nm (reproduced from ref. 17).

Figure 5.Difference infrared spectra of 8'-apo-β-caroten-8'-al in chloroform solution measured with (a) 1PE at 405 nm, (b) 2PE at 1275 nm, in addition to (c) the simulated difference (cationneutral) infrared spectrum. The zero absorbance level of each spectrum is shown as a dotted line (reproduced from ref. 17).

Branching Relaxation Pathways in the S1 State. Figure 6 shows excited-state kinetics of 8'-apo-β-caroten-8'-al in chloroform observed with mid-infrared and visible probe pulses following 1PE and 2PE. As shown in Figures 6(a) and 6(b), this long-lived species shows almost identical kinetics both in the infrared (∼1510 cm−1; 15 and 140 ps growth19) and visible (760 nm; 10 and 166 ps growth18) wavelengths. It seems that this long-lived species forms as the S1 state decays (∼17 ps from the infrared S1 bands with 2PE) but this long-lived species is formed with a slower kinetics (∼150 ps) than S1 decay. It is considered that the second minimum of the S1 state may decay slower than the S1 minimum and the long-lived species in the ground state is formed as the second minimum of the S1 state decays. The 760 nm transient absorption kinetics following 1PE at 405 nm (in Figure 6(c)) both shows a 17 ps decay (S1 state) and a 155 ps growth. It is thus confirmed that the kinetics and spectra of the long-lived species observed at 1510 cm−1 and 760 nm with 2PE and excess energy 1PE are identical.

Figure 6.Kinetic traces for (a) the long-lived absorption band at 1510 cm−1 and (b) the transient absorption band at 760 nm of 8'- apo-β-caroten-8'-al in chloroform measured with 2PE at 1275 nm. (c) The transient absorption band at 760 nm obtained with excess energy 1PE at 405 nm is shown for comparison.

When the S2 state of 8'-apo-β-caroten-8'-al is excited with 1PE with an excess vibrational energy, a branching relaxation into two minima of the S1 state is observed in both infrared and visible transient absorption measurements. Details of the branching relaxation pathway are not yet known but the following ones might be one of possible explanations. Firstly, as the S1 state of this carotenoid possesses more than one minima (possibly as a result of the conformation isomerism), the S2 state might also be composed of more than one minima. The second minimum of the S2 state, if exists, is not observed from the steady-state absorption measurements, but it could also be explained by the forbidden transition from the ground state due to the symmetry. When an extra vibrational energy (about 4200 cm−1) higher than the energy barrier between these minima is available in the S2 state, then a new relaxation pathway between the second S2 minimum and the second S1 minimum in the potential energy surfaces could occur. The second minimum of the S1 state then forms a long-lived species in the ground state, which outstays 1 ns optical delay time.

Otherwise, the S2 state of 8'-apo-β-caroten-8'-al converts to the S1 state as usual. However, the S2 decays in an ultrafast time scale (∼100 fs23) so the extra vibrational energy in the S2 state might not fully dissipated during the internal conversion into the S1 state. Then an excess vibrational energy could still exist in the S1 state, which might make the second minimum of the S1 state partially occupied and form the long-lived species in the ground state forms as the second minimum decays.

Nature of the Long-lived Species. A series of experiments on 8'-apo-β-caroten-8'-al and 7',7'-dicyano-7'-apo-β-carotene show that the S1 states of those polar carotenoids have more than one minima and that the relaxation from the second minima which are preferentially occupied by 2PE and excess-energy 1PE results in a long-lived species in the electronic ground state. Nature of the long-lived species is of great interest in unveiling the complex excited-state dynamics of these carotenoids. Evidences from infrared and visible transient absorption measurements and the DFT simulations of the infrared spectra have been considered.

As shown in Figures 2(b) and 3(b) (and also compared with a simulated difference spectrum in Figure 5), the longlived species of both carotenoids shows a very strong infrared band around 1505-1510 cm−1 (asymmetric ν(C=C)) compared to other ν(C=C) bands in the spectra in the S1 and S0 state. This long-lived species also shows a visible transient absorption band centered at 760 nm for the carotenoid 8'- apo-β-caroten-8'-al, which is generally regarded as the absorption band of the radical cations. The transient absorption band at 760 nm is blue-shifted from the D0 → D2 absorption band of 8'-apo-β-caroten-8'-al at 845 nm by the electrochemical oxidation27 and at 827 nm by the chemical oxidation with I2 and FeCl3,18 which is explained with the formation of a charge transfer complex between a carotenoid molecule and solvent molecules. The details of the cation formation in the polar solvents are not fully understood but the infrared and visible absorption band intensities for the long-lived species show a linear dependence on the carotenoid concentration (∼1 mM in 2PE and down to 75 μM in excess-energy 1PE) and show a strong dependence in kinetics on the solvent polarity.17-19

Aside from the experimental evidence, quantum mechanical calculations of the infrared spectra of both carotenoids based on the DFT have been performed to find a “good” match with experimental results.18 The ground state potentialenergy surface of both carotenoids in the coordinates of cistrans isomerization along the C13=C14 bond and a beta-ring rotation along the C6-C7 bond have been calculated by using the B3LYP functional and the 6-31G(d) basis set, but none of isomers related to these conformational changes show similar infrared spectra as Figures 5(a) and 5(b) with a very strong ν(C=C) band around 1500 cm−1. Cation and neutral radicals of both 8'-apo-β-caroten-8'-al and 7',7'-dicyano-7'- apo-β-carotene have been used for the geometry optimization and vibrational analysis and it has been found that infrared spectra of cation radicals show a very strong band around 1500 cm−1. As shown in Figure 5(c), the difference infrared spectrum between cation radical and neutral molecule of 8'-apo-β-caroten-8'-al is well matched with the measured transient infrared spectra in 2PE and the excess energy 1PE.

Figure 7.Visible transient absorption spectra of 8'-apo-β-caroten- 8'-al in (top) n-hexane and chloroform (bottom) with an excitation of 490 nm.

Formation of cation radicals in the electronic ground state and stable charge-transfer complex with a solvent molecule have been suggested for a reasonable explanation for the transient infrared and visible absorption measurements for the carotenoids 8'-apo-β-caroten- 8'-al and 7',7'-dicyano-7'- apo-β-carotene in a number of solvents

Visible Transient Absorption Measurements: Solvent Dependence. Figure 7 shows visible transient absorption spectra of 8'-apo-β-caroten- 8'-al in n-hexane (nonpolar) and chloroform (polar) solutions with an excitation of 490 nm. Transient absorption spectra of 8'-apo-β-caroten- 8'-al show the ground state bleach at 485 nm and three excited state absorption centered at 512, 540, and 620 (680) nm in n-hexane. In chloroform, the ground state bleach band appears up to 520 nm (as expected in the steady state absorption spectrum), and the excited state absorption bands appearing at ∼538, 575, and 660-680 nm become broader and red-shifted.

Considering the ultrashort lifetime of the bright S2 state, the major excited state absorption bands mentioned above would originate from the S1 state. The main excited-state absorption band at 545 and 575 nm in n-hexane and chloroform, respectively, represents the S1 → SN transition and a smaller band at 620 (and 680) and 660-680 nm in n-hexane and chloroform, respectively, represents the S1 → S3 transition. 16 The S1 → S3 transition is symmetry-forbidden in a symmetric carotenoid which belongs to C2h point group but it is observed in a polar carotenoid 8'-apo-β-caroten-8'-al (Cs point group without a C2 axis) even in a non-polar solvent (n-hexane). In polar solvents (also in acetonitrile although not shown), the symmetry breaking becomes larger so the relative intensity ratio of the S1 → S3 transition is comparable to that of S1 → SN transition.

Figure 8.Kinetic traces and their exponential fits of the transient absorption measurements of 8'-apo-β-caroten-8'-al in n-hexane with 490 nm excitation at (a) 485 nm, (b) 512 nm, (c) 545 nm, and (d) 620 nm.

The exact nature of blue-shifted excited-state absorption bands appearing as a separate band at 512 nm in n-hexane and as a shoulder band at ∼538 nm in chloroform is not clear, but this band has been assigned presumably to S*/hot S1 band.11,12 There have been debates over the intermediate dark state between the S2 and S1 state of many carotenoids10-12,28 but it lacks more experimental and theoretical evidences to unveil the exact nature of the S*/hot S1 state. We also found that the intensity of the S*/hot S1 absorption band increased 2-3 times when an excess-energy excitation of 405 nm was used instead of a 490 nm excitation.

Figures 8 and 9 show kinetic traces of the ground state bleach and three excited-state absorption bands of 8'-apo-β-caroten-8'-al in n-hexane and chloroform, respectively, and fit results by using an exponential function convoluted with the Gaussian instrumental function. The lifetime of the S2 state for both carotenoids can be measured indirectly by measuring how fast the excited state absorption from the S1 state increases probed at 545 and 575 nm in n-hexane and chloroform, respectively (see Figures 8(c) and 9(c)). The S1 → SN band shows a 1.1 ps rise in n-hexane and a faster 0.4 ps rise in chloroform solution, which are considered as the S2 lifetime. As well expected by the charge transfer character of the S1 state or the existence of the coupled charge transfer state S1/ICT especially in polar solvents, the S1 lifetime of 8'-apo-β-caroten-8'-al (25 ps in n-hexane) gets shorter (19.5 ps in chloroform; 8.0 ps in acetonitrile, data not shown). The ground state bleach and the excited state absorption bands share one lifetime, which can be considered as the S1 lifetime of the carotenoid. In the S1 → S3 transition at 620 nm in n-hexane and the S*/hot S1 transition at ∼538 nm in chloroform, a faster decaying component of 0.3-0.4 ps than the S1 lifetime have been identified, which might be understood as the time for the vibrational relaxation or vibrational cooling in the S2 and S1 states although we have not found any evidence that this process is clearly separated from the S2 or S1 decay processes. Further experimental evidences, for example, excitation dependence in the excited state dynamics of these carotenoids will be sought to solve the puzzles in the singlet excited states of carotenoids.

Figure 9.Kinetic traces and their exponential fits of the transient absorption measurements of 8'-apo-β-caroten-8'-al in chloroform with 490 nm excitation at (a) 485 nm, (b) 538 nm, (c) 575 nm, and (d) 680 nm.

 

Conclusion

Carotenoids show exceedingly complex dynamics in the singlet electronic excited states. Since carotenoids have many roles in the natural photosynthesis, understanding their excited-state dynamics and energy transfer in photosynthetic complexes might be of great interest. In this paper, we have reviewed the recent experimental and theoretical results on two polar carotenoids, 8'-apo-β-caroten-8'-al and 7',7'-dicyano- 7'-apo-β-carotene and reported the recent transient absorption measurements on 8'-apo-β-caroten-8'-al with 490 nm excitation in several solvents. Although the excited state dynamics between three singlet states (S2, S1, and S0) have been known better by various types of experiments and simulations, existence and the role of the intermediate states between the S2 and S1 or the excited-state dynamics originating from the conformational minima on the S1 potential energy surface are not yet clear.