Accepted Manuscript
Excited-State Proton Transfer of Photoexcited Pyranine in Water Observed by
Femtosecond Stimulated Raman Spectroscopy
Fangyuan Han, Weimin Liu, Chong Fang
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http://dx.doi.org/10.1016/j.chemphys.2013.03.009
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Please cite this article as: F. Han, W. Liu, C. Fang, Excited-State Proton Transfer of Photoexcited Pyranine in Water
Observed by Femtosecond Stimulated Raman Spectroscopy, Chemical Physics (2013), doi: http://dx.doi.org/
10.1016/j.chemphys.2013.03.009
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Excited-State Proton Transfer of Photoexcited Pyranine in Water
Observed by Femtosecond Stimulated Raman Spectroscopy
Fangyuan Han, Weimin Liu, and Chong Fang*
Department of Chemistry, Oregon State University, Corvallis, OR 97331 U.S.A.
*Corresponding author. Tel.: +1 541 737 6704.
E-mail address: Chong.Fang@oregonstate.edu (C. Fang).
ABSTRACT
We use femtosecond stimulated Raman spectroscopy (FSRS) to illuminate the
choreography of intermolecular excited-state proton transfer (ESPT) of photoacid
pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid, HPTS) in water. The multidimensional
reaction coordinate responsible for photoacidity is revealed to involve sequential
activation of characteristic skeletal motions during the ca. 1 ps preparation stage
preceding ESPT. The initial ring-coplanarity breaking follows in-plane ring breathing
(191 cm-1), and is facilitated by HPTS ring wagging (108 cm-1) and ring-H out-of-plane
motions (321, 362, 952 cm-1), which largely decay within ~1 ps. ESPT then occurs with
intrinsic inhomogeneity via various number of intervening water molecules over
relatively larger distances than those in acetate-water system. The intricate relationship
between the time-resolved excited-state vibrational modes of HPTS reveals the essential
role of coherent low-frequency skeletal motions gating ESPT, and the multi-staged
proton-transfer process having the kinetic isotope effect (KIE) value of 3–4 in aqueous
solution on the 5–200 ps timescale.
Keywords: Femtosecond stimulated Raman spectroscopy; excited-state proton transfer;
molecular conformational dynamics; low-frequency skeletal motions; hydrogen bond
dynamics; photoacid
1. Introduction
Proton transfer (PT) in water plays a ubiquitous and important role in many
chemical and biological processes [1-4], particularly concerning the omnipresent
hydrogen bonds (H-bonds). In living systems, protons catalyze a myriad of aqueous
reactions and serve as an important means for transient energy transport and storage, so
the significance of investigating proton functionality cannot be overstated. A widely used
experimental approach to study PT is to precisely activate it via photoexcitation of a
photoacid. For these molecules, absorption of a photon leads to a significant increase of
the molecular acidity, and therefore triggers a series of events contributing to the overall
processes of intermolecular excited state proton transfer (ESPT) [3]. These molecular
processes can be distinguished as electronic redistribution and hydrogen bond
rearrangement in sub-femtosecond (sub-fs) to fs time range, proton dissociation and
1
solvation processes in sub-picosecond (sub-ps) to ps time scale, and proton diffusion as
well as proton recombination and quenching reactions in ps to nanosecond (ns) time
regime. The multidimensionality of the potential energy surface (PES) of photoacids
needs to be studied in considerable detail to reveal the anharmonic coupling matrix
within the molecule, and the structural origin of chemical reactivity [5-10].
Pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid, or HPTS), a commonly used
photoacid, has a pKa value of ~7 in the electronic ground state (S0) that drops to ~0 upon
photoexcitation [11-27] and serves as a pragmatic system to scrutinize the reaction
mechanism for a transducing photosensitive molecule undergoing ESPT. HPTS has
strong photoabsorption near 400 nm and can thus be easily triggered through optical
excitation, which induces a vertical transition between S0 and the electronic excited states
(commonly the singlet excited state S1), with the subsequent ESPT followed by
fluorescence and other electronic absorption changes. The vibronic wavepacket generated
by the optical excitation samples the excited-state PES to relax energy, and eventually
finds ways to go back to S0. The rate and mechanism of intermolecular ESPT of HPTS in

water (e.g., initial step ROH*+H2O→RO *+H3O+) have been monitored with different
electronic and vibrational spectroscopies, including the time-resolved fluorescence
spectroscopy [11-13, 15], transient visible [18, 22] or mid-IR spectroscopy [17, 19-21,
23-26]. In particular, Tran-Thi, et al. and Leiderman with co-workers used both timeresolved fluorescence and transient absorption spectroscopy to investigate ESPT from
HPTS to water [15, 18]. They found that the ESPT processes involve ultrafast solvation
dynamics (0.3–0.8 ps), two steps of proton dissociation (the formation of a contact ion


pair RO *∙∙∙H3O+ in ~3 ps and separation of ion pair RO *----H3O+ in 87–100 ps), and a

subsequent long-lifetime RO * emission/fluorescence step (~5.3 ns). In D2O, the
dynamics of the two proton-dissociation steps are delayed to ~5 ps and 210–300 ps,
respectively [17-19, 22, 24]. This represents a kinetic isotope effect (KIE) of ca. 3 for the
long time constant observed in the ESPT dynamics [3].
Recently, ESPT of HPTS and different carboxylate base complexes dissolved in
aqueous solution was extensively studied using fs UV-pump mid-IR-probe spectroscopy
[17, 20, 21, 23-25], wherein the PT reactions from HPTS to the base exhibit multiexponential decay processes with sub-ps to hundreds of ps time constants. Upon addition
of stronger bases such as acetate to aqueous solution, the ESPT dynamics speed up
significantly due to the intermolecular PT from HPTS to the base molecule, which forms
a greater amount of more tightly bound H-bonding complexes over relatively short
distances, and dominates the reaction through the water medium. It was reported that the
optimal PT distance is ~0.75 nm, corresponding to approximately two water molecules in
between the proton donor (acid) and acceptor (base) in the Eigen-Weller model [1, 21,
25]. A recent theoretical study on the ESPT dynamics of 6-hydroxyquinolinium (6HQc),
a photoacid smaller than HPTS, found that the threshold of the water cluster size is 3 for
its ESPT in aqueous solvent [28]. Water participates in the PT reaction by receiving a
proton from the proton-donating group and then releasing a proton to the protonaccepting group in the sequential or concerted manner depending upon the base
concentration [23, 25]. In protic solvents such as water, a single H-bond or a H-bonded
water matrix connects the proton donor and accepter, in which protons are highly mobile
and can be transferred via intricate energy relaxation pathways. In addition, water will
2
stabilize and/or destabilize various charge-transferred species as PT occurs. However,
after years of active research on ESPT from HPTS to water, the mechanism still remains
unclear particularly concerning the involved conformational dynamics of the photoacid.
To examine the ESPT process in solvated HPTS is to understand its
photochemistry using a direct bottom-up approach. We need to go beyond measuring the
electronic response of the molecular system or collecting the time-resolved fluorescence
data, because those data can only infer the electronic PES with no detailed vibrational or
structural information. The visible-pump mid-IR probe studies have their shortcomings
including the limited time resolution and spectral range, and some uncertainty correlating
relatively broad spectral features with specific molecular motions. In order to capture the
molecular snapshots of the photoexcited HPTS immediately following photoexcitation
and to track the choreography of the transferring proton in conjunction with all the
coupled atomistic motions, the time-resolved vibrational spectra of HPTS on S1 need to
be analyzed in detail. The ability to concomitantly track the acceptor response is an added
bonus [27]. The emerging femtosecond stimulated Raman spectroscopy (FSRS) [29-31]
provides that level of information with simultaneously high spectral and temporal
resolution, perfectly suitable to probe the non-equilibrium atomic motions of photoacids
following photoexcitation. Since fluorescence is typically on the ns timescale, the fs
resolution of the FSRS setup ensures the unravelment of earlier (i.e., pre-fluorescence)
processes, mainly skeletal motions and ESPT, following the vibronic wavepacket motion
out of the Franck-Condon (FC) region of ROH* (PA*) toward the deprotonation barrier

crossing, and before the nascent RO * (PB*) state relaxes back to PB via fluorescence.
One of the most appealing advantages of FSRS is the resolution achievable in
both the spectral and time domain, which merits some discussion. This is accomplished
by the unique sequence of laser pulses with either the fs or ps time duration, and the
dispersive signal detection scheme without temporally resolving the Raman scattering
photon carrying the impulsively excited vibrational coherence free induction decay (FID)
information. The spectral resolution is thus determined by the FID time as well as the
Raman pump pulse duration, because the Fourier transform of the coherence decay
convoluted with the Raman pump pulse duration will dictate the frequency linewidth of
the observed vibrational mode. The typical FID time of a vibrational coherence is on the
timescale of 1–3 ps, and since we can generate the Raman pump with ~4 ps full-width-athalf-maximum (fwhm) [27], the spectral resolution can be well below 10 cm-1. The
temporal resolution, on the other hand, is decoupled from the pulse duration of the
Raman pump and can be smaller than 30 fs. This is due to the fact that the vibrational
coherence is precisely generated upon the simultaneous arrival of the Raman pump and
probe photons on the sample spot, and because the Raman probe is a broadband pulse
with ~30 fs pulse duration, its cross correlation with the preceding fs actinic
photoexcitation pulse is well defined and can be very small. It is therefore key to have
that fs-ps-fs pulse sequence to enable us precisely trigger vibrational coherences and
probe structural evolution in real time [31, 32]. The ability to track non-equilibrium
conformational dynamics thus makes FSRS a unique and powerful technique to study
photosensitive molecules in condensed phase. Furthermore, the advantages of FSRS
include the broad spectral window spanning more than 1200 cm-1 for one spectrograph
grating position, selective excitation of the chromophore (e.g., it could be embedded
inside a big protein such as wild-type green fluorescent protein, a.k.a. wtGFP) [9],
3
convenience of using water as the solvent, fluorescence rejection, moderate sample
concentration requirement, fast data acquisition (e.g., the CCD detector is synchronized
with the main laser repetition rate of 1 KHz) with high signal-to-noise ratio (SNR), and
versatile control of each individual pulse across the UV to NIR regime [27, 33].
In our previous report [27], we used the newly developed FSRS setup [29-31, 34]
to capture the molecular structural snapshots of HPTS in S1 as it pushes the hydroxyl
proton toward the acetate ion in aqueous solution. The temporal evolution of excited-state
Raman modes, especially in the low-frequency regime, reveals the remarkable ultrafast
structural dynamics events along the ESPT multidimensional reaction coordinate of
photoexcited HPTS starting from time zero. The corresponding vibrational frequency
range of 100−500 cm−1 cannot be directly observed in the mid-IR region in water. The
molecular skeletal motions therein reduce the distance between the proton donor and
acceptor, which may adopt a largely different electronic character between S0 and S1
states, and result in lowering the effective barrier to proton hopping [27, 35-39]. Our
experimental results show that the vibrational marker bands attributed to the deprotonated
form of HPTS appear earlier and faster than the nascent monomeric acetic acid peak,
indicating that various number of water molecules actively participate in the ESPT chain.
The goal of this contribution is to delineate the choreography of intermolecular
ESPT reaction of photoexcited HPTS in pure water. The absence of strong bases such as
acetate excludes the complexity of various degrees of driving forces to attract the
hydroxyl proton of HPTS, and provides a clean background to investigate the bilateral
interaction between HPTS and the H-bonded water matrix. Upon photoexcitation of
HPTS, water will act as the proton acceptor because its pKa is higher than zero (the pKa
for the S1 state of HPTS). But since the proton-accepting strength of water is weaker than
that of acetate, slower ESPT dynamics may be observed. Also, the accompanying skeletal
motions of HPTS that become Raman active and show pronounced activities should be
exposed by the time-resolved FSRS spectra of the S1 state of HPTS surrounded by water
molecules only. The comparison with previous results on HPTS in acetate water solution
will provide deep insights on the dynamic variation of the H-bonding network and the
driving force for intermolecular ESPT, as well as the conserved skeletal motions gating
ESPT between photoexcited HPTS and proton acceptor molecules in aqueous solution.
2. Experimental methods
The photoacid pyranine (HPTS) was purchased from TCI America (≥85%). It was
previously found to have no detectable spectral difference from the ≥97% purity sample
from Aldrich, so we used the ≥85% purity sample (11 mM) in millipore water solution
(pH≈6) after 0.22-m filtration but without further purification [27]. The Raman spectra
of HPTS through the ESPT reaction are collected in both H2O and D2O (99.9% D,
Cambridge Isotope Laboratories, Inc.) solutions. Though we did not intentionally use
DPTS to start with, the concentration difference between HPTS (11 mM) and D2O (~55.3
M) leads to the almost complete deuteration at the phenolic hydroxyl end of HPTS after
1-2 days of storage and H/D exchange in solution at room temperature. The UV/Vis
spectrum shows OD≈26/mm at the protonated HPTS (PA) absorption peak (PA≈24,000
M-1·cm-1) and this is to ensure that we have enough SNR for both ground-state and
4
excited-state FSRS data. The S0 and S1 PES of PA dictates the electronic resonance
conditions here and the 800 nm Raman pump pulse we use does not induce strong Raman
vibrational features, in contrast to the previously studied wtGFP chromophore case [9].
The experimental procedure has been reported in detail elsewhere [27, 34].
Briefly, our FSRS setup (Fig. 1) uses a fs Ti:Sapphire laser amplifier system (Legend
Elite USP-1K-HE, Coherent Inc.), which provides ~35 fs, 4 W laser pulse centered at
800 nm with 1 kHz repetition rate. Half of the output laser beam is split into three beams
required for FSRS, which are depicted in different colors in Fig. 1. Around 200 μJ/pulse
of the laser output is frequency-doubled using a β-barium borate crystal (BBO, type-I,
phase matching angle θ=27.8°, 0.3 mm thickness) to generate the actinic pump pulse at
400 nm with the pulse energy of 50 μJ/pulse, then compressed by a prism pair (Suprasil1, CVI Melles Griot) to ~40 fs. The average power of the 400 nm photoexcitation pulse
is then attenuated to ca. 1 mW for excited-state FSRS measurement, to ensure a stable
spectral baseline as well as enough ground-state (S0) depletion to observe excited-state
vibrational features of HPTS in water in the linear regime [34]. Persistent and unchanged
S0 depletion is observed as spectral dips at S0 vibrational frequencies when higher
photoexcitation power (e.g., 1.5 mW) is used, whereas lower power at 0.5 mW yields
smaller depletion than the 1 mW photoexcitation case. About 15 μJ/pulse of the laser
output is focused on a Z-cut single crystal sapphire plate with 2 mm thickness to generate
the supercontinuum white light. The wavelength range from 805–915 nm of the white
light that corresponds to ca. 100–1600 cm−1 Stokes Raman shift to the 800 nm
fundamental is selected using a long-wavelength pass filter, and then compressed by a
fused silica prism pair (Thorlabs, Inc.) to produce ~35 fs broadband Raman probe pulse.
The Raman pump pulse with ~10 cm−1 bandwidth and pulse duration of 3.5 ps is
produced by a homemade grating-based spectral filter (1200 grooves/mm, wavelength
first order at 750 nm, blaze angle θ=26.7°). The collimated Raman pump, probe, and the
photoexcitation beams are all focused onto the sample cell using an off-axis parabolic
reflective mirror (to avoid chirp), with the focus size of ~150 m for the Raman pump
and actinic pump beams, and ~100 m for the much weaker Raman probe beam.
The dispersed FSRS spectra are first calibrated using carbon tetrachloride (CCl4)
and ethanol (CH3CH2OH) mixed solution as a standard that spans the Raman frequency
range from ca. 200–1600 cm−1 [27]. The ground-state FSRS signal of the standard
solution is maximized through spatial and temporal overlap adjustment of the Raman
pump and probe beams, which are at a crossing angle of ~3.5º to ensure a relatively long
interaction length. The average power of the 800 nm Raman pump pulse is ~6 mW and
we set it to balance the signal strength, the peak width and the stability of the baseline. A
higher Raman pump power will increase the signal strength but at the expense of broader
peaks and fluctuating baselines particularly in the low-frequency region. The probe beam
after the sample is sent into a spectrograph and dispersed by a 600 grooves/mm grating
(wavelength first order at 1000 nm, blaze angle θ=17.5°), and then imaged onto a frontilluminated charge-coupled device (CCD) camera (Princeton Instruments, PIXIS 100F)
consisting of a 1340 × 100 pixel array, synchronized with the main laser repetition. The
Raman pump beam is chopped at half of the laser repetition rate (i.e., 500 Hz) to measure
the single-shot Raman probe spectrum, with the sequential Raman pump on and off
conditions repeatedly. The FSRS spectrum is collected, calculated, averaged and
displayed on-screen using an updated LabVIEW program incorporating STIK scientific
5
imaging toolkit (R-Cubed software, NJ) for the CCD camera. The spatial and temporal
overlap between the two fs pulses, the 400 nm photoexcitation and the Raman probe
continuum at a crossing angle of ~4º, is finely adjusted by optimizing the Optical Kerr
Effect (OKE) signal at the front portion in the liquid standard sample, with the typical
cross-correlation time measured to be ~140 fs full width at half maximum (fwhm) for our
time-resolved FSRS experiments in the aqueous solution. The sample volume of ~800 µL
is used in a 1-mm pathlength flow cell (48-Q-1, Starna Cells) to avoid sample
degradation under intense pulsed laser excitation. Higher excitation power at ~1.5 mW
causes irreversible photodegradation despite rapid flow of the sample solution, showing
strong ground-state vibrational peak depletion even when the 400 nm photoexcitation
pulse arrives later (e.g., 3 ps) than the Raman pump-probe pair. Less than 5% population
change (mainly from the protonated to the deprotonated form of HPTS) is observed from
the UV-Vis spectra before and after ~2 hours of excited-state FSRS measurement with 1
mW photoexcitation power used, so the population variation effect on our FSRS spectral
analysis is negligible. The kinetics of the stimulated Raman intensities in the excited-state
FSRS spectra (see below) are multi-exponentially fitted and convoluted with the
aforementioned instrument response time of ~140 fs.
3. Experimental results
The ground-state FSRS spectrum of HPTS has been measured at two grating
positions of the spectrograph (Fig. 1), in order to cover the wide spectral range of 50–
2000 cm-1. The slit width in Raman pump generation is set below 0.1 mm still with
enough output power to induce the Raman transition, enabling the observation of the
narrow linewidth of the Raman peak close to the natural linewidth seen in the continuous
wave (cw) excitation case. Fig. 2 shows the FSRS spectrum of the S0 state of HPTS, with
the vibrational modes above 1000 cm-1 dominating the spectrum. It is notable that the
maximum electronic absorption peak of HPTS in water is at ~404 nm for the protonated
form (PA) of the photoacid [27], therefore we rely on the two-photon absorption cross
section to obtain the ground-state FSRS spectrum of HPTS with the ~800 nm Raman
pump pulse. The weaker spectral features observed in the excited-state FSRS spectra of
HPTS in water in comparison to the wtGFP chromophore data [9] suggest that the
resonance enhancement factor for S1 vibrational features is insignificant for HPTS
because the 800 nm Raman pump does not closely match the energy gap between S1 and
other higher-lying electronic states of the PA or PB form [40, 41].
Density functional theory (DFT, RB3LYP and UB3LYP) calculations using
Gaussian 09 [42] and the 6-311G++(2d,2p) basis sets on the protonated HPTS molecule
(–3 charge, singlet state), with the water solvation effect included by a polarizable
continuum model (PCM) using the integral equation formalism (IEF-PCM-H2O), yield
Raman peak frequencies to be compared with experimental results using a typical scaling
factor of 0.96. The calculated ground-state frequencies are considered to be more
accurate in the low-frequency region due to the collective nature of those participating
atomic motions, which should remain largely unshifted in the first singlet electronic
excited state S1 [9, 39]. The detailed assignment of key vibrational modes that show
significant activities in H2O is listed in Table 1, and can also be found in our earlier
6
report [27]. In brief, the skeletal modes below 1000 cm-1 assume weak Raman activities
in the ground state due to the coplanarity of the aromatic four-ring system of HPTS,
which agrees with DFT calculation results. The ring-H rocking, C–O stretching and C=C
stretching motions above 1000 cm-1 exhibit strong peak intensities due to their relatively
high tendency for electron redistribution and large Raman polarizabilities upon electronic
excitation [40, 41]. Reckoning the two-photon absorption of HPTS at the 800 nm Raman
pump wavelength, it suggests that these high-frequency modes are along the PES slope in
the FC region of PA* with the bottom well of S1 displaced from the equilibrium position
of S0. It is notable that the deprotonated (PB) HPTS has relatively higher intensities of the
low-frequency modes in comparison with the protonated (PA) HPTS, consistent with a
more twisted structure of the chromophore after the departure of the phenolic proton [27]
even in S0. A pictorial representation of the ESPT process from HPTS to water through
the H-bonding chain can be found in the Fig. 2 insert.
Fig. 3 shows the time-resolved FSRS spectra of HPTS in water following ~1 mW
of 400 nm photoexcitation pulse, from –1 ps to 150 ps. The relatively high peak power is
used for excitation due to the high concentration of HPTS being used (OD≈26/mm at the
PA absorption peak), making it essentially nontransparent to the 400 nm laser pulse. The
local heating effect is minimized by rapidly flowing the sample through a sealed reservoir
away from light at room temperature. The ground-state (S0) Raman spectrum is collected
periodically throughout the excited-state FSRS measurement and averaged, followed by
subtraction from each time-delayed spectrum to discern the difference spectrum with
various dips and peaks, then a percentage of the fitted S0 spectrum is added back to
consistently fill those dips and reveal the positive excited-state features across the wide
spectral detection window. The negative-time spectra corroborate the robustness of the
current experimental approach in that no chemistry is happening before photoexcitation,
and the sample also holds well without photodegradation throughout the FSRS scan. The
maximum ground-state depletion achieved in this experiment is ~12%. It is possible that
individual vibrational modes might assume slightly different bleaching dynamics, but to a
generally good approximation, we envision the ground-state vibrational peaks to be
depleted simultaneously upon reaching a different electronic state. Though we commonly
attribute the remaining positive peaks to S1 vibrational transitions, it is conceivable that
these transient features might be associated with S0 vibrational transitions [9, 43]. The
latter case requires the photoexcited wavepacket to be directly generated on S0, or for the
initially generated S1 wavepacket to quickly relax back to S0 on the timescale of our
FSRS measurement, which is fs to ps, thus excluding the fluorescence pathway that is
typically on the ns timescale. However, the fact that the excited-state spectrum at T=0 fs
differs from the ground-state spectrum and also shows wider linewidth indicates that the
vibronic wavepacket and vibrational coherence is generated on S1, with significantly
different electronic distribution over the HPTS ring system. Furthermore, the pronounced
activities of the low-frequency modes following photoexcitation correlate well with the
spectral activities of the high-frequency modes, with the peak kinetic analysis intimately
reflecting the ESPT dynamics responsible for its photoacidity in solution phase.
There are a number of prominent low-frequency modes in Fig. 3 that show PA*
characteristics, which grow in following photoexcitation, and gradually decay away.
These include 108, 125, 143, 191, 321, 362, 630, and 952 cm-1 (Table 1), which exhibit
different dynamics that will be analyzed and discussed in detail below. The correlated
7
high-frequency modes appear at 1048, 1180, and 1285 cm-1. In particular, the vibrational
modes at 143, 191, 952, and 1180 cm-1 are predominantly active before ~1 ps, and are
labeled in red in Fig. 3 and also highlighted by red boxes. Evidence of quantum beats
shows up in the kinetic plot of the 143 cm-1 mode intensity. The 870 cm-1 mode shows
complex dynamics within the experimental time window of 150 ps, and involves ringhydrogen out-of-plane (HOOP) motions from both the PA* and PB* modes as shown by
calculations. The mode at 1138 cm-1 is an interesting case in that the ephemeral feature
before 1 ps might be closely associated with PA* adopting different electronic character
from the ground state in association with photoacid skeletal motions, while the gradual
increase of the peak intensity after 1 ps can be attributed to the accumulation of PB* as
ESPT occurs. The modes at 276 and 460 cm-1 have a delayed onset in comparison to the
promptly emerged PA* modes, and display the gradual increase within the temporal
window of the experiment, so we assign them to PB* modes (labeled in blue in Fig. 3)
that will eventually decay away on the ns timescale due to fluorescence.
Fig. 4 displays the temporal evolution of the 1048 cm-1 mode that is characteristic
of the PA* dynamics up to 150 ps. The integrated peak area is used to best capture the
essence of the stimulated Raman peak intensity. After deconvolution from the crosscorrelation time of 140 fs measured from the OKE signal of the actinic pump and Raman
probe pulses, the 1048 cm-1 mode rises within ~210 fs, and decays bi-exponentially with
time constants of 4.5 ps (32%) and 150 ps (68%). Fitting results of various vibrational
modes are listed in Table 1, and the relative weight of the fitted exponentials is shown in
parentheses. The 150 ps time constant is less accurately determined than the 4.5 ps time
constant due to the detection time window, limited by the 1-inch motorized translation
stage that we used. The insert in Fig. 4 shows the detailed time-dependent peak intensity
plot up to 10 ps, with the signal strength indicated by the double-arrowed vertical line.
The time correlation between the nascent PA* and PB* modes can be found in
Fig. 5. The 1285 cm-1 PA* mode rises exponentially with a time constant of ~190 fs, and
decays with two time constants of 4.3 ps (40%) and 215 ps (60%). Note that the long
decay time constant is larger than that of the 1048 cm-1 mode, which may hint an
overlapping PB* mode contribution around 1285 cm-1. The 460 cm-1 PB* mode rises
biexponentially with time constants of 680 fs (26%) and 130 ps (74%); while the 1138
cm-1 PB* mode after 1 ps can be fitted by two rising exponentials with time constants of
5 ps (12%) and 78 ps (88%). The difference between the two PB* modes can be
explained by the atomistic motions responsible for them: the 460 cm-1 mode is primarily
an HPTS ring asymmetric wagging motion and may start gaining Raman intensity in an
earlier stage of ESPT, whereas the 1138 cm-1 mode is the HPTS phenolic CO···(H) inplane rocking motion that takes longer to appear but rises faster on the tens of ps
timescale overall (78 ps vs. 130 ps). The sub-ps onset of the 460 cm-1 mode is indicative
of the activation of some coherent low-frequency modes preceding ESPT, in which the
ring wagging motion plays an important role in modulating the intermolecular distance
between the hydroxyl group of HPTS and the neighboring proton acceptors, e.g., water
molecules in this work. The transient coherent PA* ring wags can potentially generate a
small proportion of (~26% if estimated from the fast rise component of the 460 cm-1
mode) PB* state, with its ring wagging mode on the rise with a 680 fs time constant that
is delayed to some of the fastest PA* in-plane modes (e.g., the 191 cm-1 mode).
8
A collage of low-frequency modes of photoexcited HPTS is plotted in Fig. 6, and
their time-resolved peak intensity analysis is very informative. The most transient PA*
peak, the 191 cm-1 mode, shows a rising exponential time constant of 320 fs and a decay
constant of 540 fs. The 321 cm-1 PA* mode rises with a time constant of 650 fs, and has a
biphasic decay with time constants of 1.1 ps (80%) and 75 ps (20%). The 276 cm-1 PB*
mode intensity kinetic trace shows a biphasic rise with time constants of 550 fs (85%)
and 110 ps (15%), which has some interesting differences from the 460 cm-1 PB* mode
dynamics and will be discussed later. The peak kinetics and the frequency shift from
ground-state peaks, as well as the intricate relationship between various time-resolved
vibrational modes in FSRS, exclude the possibility of a simple scheme of Raman pump
attenuation-induced ground state depletion. The matching rise and decay time constants
such as ca. 550 and 650 fs (see Table 1) from multiple vibrational peak kinetic analysis
infer the ESPT reaction mechanism of HPTS in water, showing vivid atomistic details as
the chemical reaction proceeds on the anharmonic multidimensional PES in real time.
To further investigate the structural origin of the observed kinetic processes of
photoexcited HPTS, we have also conducted FSRS measurements on HPTS in D2O. The
S1 vibrational modes at 1048 and 1138 cm-1 in H2O redshift to 1042 and 1136 cm-1 in
D2O, respectively, which agrees with the retardation of mode vibrations upon deuteration.
It is evident from the blue (H2O) and red (D2O) traces in Fig. 7 that the PA* decay and
PB* rise all slacken in D2O, wherein the 1042 cm-1 mode rises with the time constant of
610 fs, and decays bi-exponentially with time constants of 24 ps (40%) and 650 ps (60%)
(see Table 2). This represents a KIE of ~5.3 for the short time component, and ~4.3 for
the long component of the peak intensity decay. The 1136 cm-1 mode in D2O has a
biphasic rise with time constants of 21 ps (12%) and 200 ps (88%). A detailed account on
the corresponding KIE appears below in the Discussion section. The enlarged PB* peak
intensity kinetic plot up to 10 ps is shown in the insert, manifesting the complex peak
dynamics of the HPTS phenolic CO···(H/D) in-plane rocking mode before 1 ps, which
might be affected by the aforementioned transient PA* feature in that spectral region. It is
notable that the short component of the PB* peak rise matches the short component of the
PA* peak decay (5 vs. 4.5 ps in H2O, and 21 vs. 24 ps in D2O), but the long component is
significantly shorter for the PB* rise than the PA* decay (78 vs. 150 ps in H 2O, and 200
vs. 650 ps in D2O). This can be explained by the fact that although ESPT is the dominant
energy relaxation pathway for PA* to cross the barrier and produce PB*, there might be
other relaxation pathways present to dissipate the photoexcitation energy being absorbed
by HPTS. For instance, the direct fluorescence from the PA* state to PA leads to a decay
time constant of ~4.5 ns, which can contribute to the elongation of the overall decay
dynamics of PA*. On the contrary, the initial PB* peak intensity rise is a direct
consequence of ESPT and is a more reliable parameter to report on the PT process and to
compare the rate difference upon deuteration.
The two lowest-frequency modes at 108 and 125 cm-1 observed in the timeresolved excited-state FSRS spectra in Fig. 3 are fitted with two overlapping gaussian
profiles, and plotted against the time delay in Fig. 8. The 108 cm-1 mode rises with the
630 fs time constant, and has a biphasic exponential decay of 1.1 ps (75%) and 1.7 ns
(25%). The 125 cm-1 mode has a delayed onset of ~1 ps, which can then be fitted with the
biphasic rise time constants of 650 fs (55%) and 5.3 ps (45%), along with an exponential
decay time constant of ~270 ps. Since we have observed a similar 106 cm-1 mode
9
previously in 11 mM HPTS in 2 M acetate water solution [27], and the conservative
nature of such skeletal collective motions leads to similar vibrational frequencies in
various aqueous solutions, we attribute the 108 cm-1 mode to the HPTS 4-ring out-ofplane (OOP) wagging motion of the photoexcited protonated chromophore (PA*). The
more pronounced 125 cm-1 mode that gains intensity after a dwell of 1 ps but decays
away after reaching the maximum at ~20 ps may be associated with a related ring
translational motion of PA*, which might better facilitate the later stage of ESPT (e.g.,
nascent ion-pair separation) after the initial stage (e.g., heavy atom optimization of the Hbonding chain) is largely over on the ca. 5 ps timescale.
Evidence of the coherent quantum beats can be clearly seen in the time-dependent
intensity plot of the 952 cm-1 mode (Fig. 9). The convoluted multi-exponential fitting
with the 140 fs cross-correlation time yields the rise time constant of ~140 fs, and the
biphasic decay time constants of ~600 fs (78%) and 90 ps (22%). The insert of Fig. 9
manifests the mode intensity quantum beats with a period of ~360 fs, which is prominent
up to 1 ps. Considering that this mode can be attributed to HPTS ring-HOOP motions
mixed with some in-plane ring deformation from DFT calculations (Table 1), it is
justifiable to correlate this particular PA* motion with one of the early-time reaction
coordinates to set up the stage for subsequent ESPT from HPTS to water.
4. Discussion
The ability to simultaneously monitor a multitude of vibrational modes on the
electronically excited state (e.g., S1) in real time makes FSRS a powerful structural
dynamics tool to study photosensitive molecules in solution phase [30, 31]. Here we
investigate the commonly used photoacid HPTS in pure water, without the presence of
any other proton acceptor, to ensure that ESPT only occurs between HPTS and water
molecules within the H-bonded network upon photoexcitation. The simplicity of this
system construct leads to the direct correlation between experimental data and theoretical
calculations regarding the chromophore in complex with water molecules in condensed
phase. The transient disappearance of PA* and appearance of PB* modes should in
principle be correlated with ESPT, in competition with other radiative/nonradiative
relaxation pathways from PA* or radiative pathways from PB*, on the fs to ns timescale.
Furthermore, the excited-state vibrational peak temporal evolution (Figs. 3–9) from the
well-defined time zero reveals the detailed elementary steps of molecular conformational
dynamics responsible for ESPT and the photoacidity of HPTS in aqueous solution with
unprecedented precision. It is important to point out that we are not temporally resolving
the vibrational coherence FID once it is generated by the Raman pump-probe pair
because the Raman pump pulse is ps in duration and the CCD camera keeps recording the
FSRS signal within every 1 ms at the laser repetition rate. Therefore, the vibrational
mode intensity evolution revealed in our FSRS data probes the instantaneous molecular
conformation (not the FID or vibrational population relaxation typically on the few ps
timescale) at a certain time delay after photoexcitation, and reports on (1) the coherent
nuclear motion of the molecule and/or molecular complex navigating the S1 PES from the
FC region [9, 31, 44-47] to (2) further structural evolution going beyond the vibrational
coherence decay time window. These exemplary vibrational excitations (besides
10
characteristic modes that are strongly coupled to the optical transition hence the
generation of transient vibronic wavepackets) and their temporal evolution throughout the
ESPT reaction on the fs to ps timescale thus provide vivid molecular conformational
snapshots of HPTS as it navigates the dynamic excited-state PES (varying with the
solvent rearrangement) to transfer the phenolic proton to water before fluorescence.
Excited-state FSRS spectrum of HPTS in H2O and D2O. A number of prominent
vibrational modes from 50—1450 cm-1 appear in the excited-state FSRS spectrum of
HPTS in water (Fig. 3) upon adding back a certain percentage of the multiple-gaussianfitted ground-state spectrum. There are several transient low-frequency modes below
1000 cm-1 that are either associated with PA* or PB*, and the most effective way to
assign them is by the individual kinetic analysis and Gaussian calculations (main results
shown in Table 1). Considering the typical ps timescale of ESPT in solvated systems,
excited-state vibrational features that appear promptly following photoexcitation but
decay on the ps timescale can normally be assigned to PA*, while the modes that appear
later but keep rising are possibly associated with PB*. This is due to the temporal
resolution provided by the fs photoexcitation and Raman probe pulse, and the temporal
precision of the vibrational coherence being generated and detected in excited-state FSRS
spectrum. The main reasons for the PA* vibrational modes to diminish can include the
loss of resonance enhancement as the vibronic wavepacket moves out of the FC region
(e.g., akin to the case of the wtGFP neutral chromophore [9]), or the wavepacket
movement toward another electronic state (e.g., barrier crossing from PA* to PB*), and
further structural evolution to convert more PA* to PB* population. As a result, unless
there is a mode with similar vibrational frequency to PA* and similar resonance
condition with the Raman pump pulse, the peak intensity dynamics of PA* will report on
the decay of PA* via ESPT and other energy relaxation pathways. On the other hand, the
PB* mode emerges with shifted frequency from the ground state PA modes, and will
probe the rise of PB* state via ESPT only with high specificity, because other relaxation
processes from PB* generally occur on much longer timescales.
The ~4.5 ps short decay time constant of the 1048 cm-1 mode for photoexcited
HPTS in water is longer than the ~1.6 ps decay observed in water with 2 M acetate [27],
and also with a reduced amplitude (32% in water vs. 60% in acetate water). This result
indicates that the absence of the stronger proton acceptor largely shuts off the direct
HPTS-base ESPT channel through pre-existing highly polarized H-bonds, wherein the
solvent dynamics within the first solvation shell of the photoacid dominate the ESPT
pathway on the 1–2 ps timescale. ESPT that solely relies on the HPTS-H2O H-bonding
network is more subject to the labile water molecular dynamics, and likely takes longer
time as well as distance to establish the effective proton transfer chain to accommodate
the significantly increased acidity of HPTS. Previous experiments using time-resolved
fluorescence/absorption and visible pump-probe or mid-IR probe spectroscopies [3, 15,
19, 22] have revealed comparable short decay time constant at ~3 ps and the long decay
time constant of ~90 ps for the deprotonation of HPTS in water. The latter temporal
component is close to our result here, ~150 ps for the long decay time constant of the
1048 cm-1 mode, and ~78 ps for the long rise time constant of the 1138 cm -1 mode (see
Table 1). We consider that the 1138 cm-1 mode, attributed to PB* that represents the
deprotonated state of HPTS, reports on the ESPT process more faithfully. In contrast, the
11
decay dynamics of the 1048 cm-1 mode of PA* is more susceptible to other relaxation
pathways such as the direct emission from PA* and the geminate recombination of the
separated ion pairs [13, 14], which is diffusion controlled and occurs on a typical
timescale of 300–400 ps at room temperature for the PA* population that has dissociated.
Ground state FSRS results on HPTS in D2O (data not shown) corroborate the
aforementioned peak assignment. Almost all the vibrational peaks of HPTS remain
unshifted upon solvation in pure D2O, except that the 1050 cm-1 mode redshifts to 1048
cm-1, and the 1292 cm-1 mode blueshifts to 1298 cm-1 that is associated with the
deprotonated chromophore. The kinetic analysis of the characteristic PB* mode in D2O at
1136 cm-1 shows two rise time constants of 21 ps (12%) and 200 ps (88%) (Table 2). The
relative amplitudes of the exponential fitting are the same with the two rising components
in H2O, but with significantly larger time constants. This result is reminiscent of the
ESPT process from HPTS to the solvent, pure H2O or D2O here, which reports on the
proton motion on the ps timescale and the complimentary rise of an aromatic ring
vibrational response that is characteristic of PB*. The slowing down of ESPT upon
deuteration will be quantitatively addressed below with reference to KIE.
The most significant difference of this work in comparison to our previously
reported FSRS data for HPTS in 2 M acetate water solution [27] can be appreciated from
the kinetic analysis of the observed excited-state vibrational modes. It is expected that at
thermal equilibrium and the electronic ground state (S0), water solvates the HPTS
molecules by providing a solvation shell around them, while still maintaining various Hbonding geometries with several neighboring water molecules. There is intrinsically an
inhomogeneity regarding H2O molecules in the H-bonding chain involving HPTS, i.e.,
different number of H2O molecules might be participating in the chain starting or ending
with the phenolic hydroxyl group of HPTS. It has been proposed that the Grotthuss
hopping model of the proton [3, 48] plays a dominant role in ESPT within these Hbonded reactive complexes, via different numbers of intervening H-bonded H2O
molecules, and by interchanging of the covalent and H-bonds. As a result, the proton
does not need to diffuse over large distances; rather its charge is transferred between the
donor and acceptor through the highly directional H-bonding chain. This mechanism is
supported by the previously reported KIE of 1.4–1.5 of the proton/deuteron continuum
peak dynamics at early time (<10 ps) in 10 mM HPTS, 2 M acetate solutions in H2O/D2O
[21, 24]. It is worth mentioning that the acetate ion represents a stronger proton acceptor
than water, thereby providing a significant amount of driving force to facilitate ESPT
following photoexcitation and the increased acidity of HPTS. In other words, ESPT
between HPTS and acetate can be attributed to an adiabatic PT process as the proton
moves along a preformed highly polarizable H-bond, without significant barrier tunneling
or H-bonding reorganization, and mainly on the sub-ps to ps timescale [24].
However, in pure water the observed KIE is a factor of ~5.3 for the short
component, and ~4.3 for the long component of the 1048 cm-1 PA* peak intensity decay.
We consider this KIE to lack in accuracy when compared to that from the PB* increase
(e.g., 1138/1136 cm-1 modes in H2O/D2O, with a KIE of ~4.2 for the short component,
and ~2.6 for the short component), which has to be a direct consequence of ESPT. This is
still larger than the KIE of ~1.8/2.4 derived from previous visible pump-probe
measurements on HPTS in H2O/D2O [22], but similar to ~2.8 for the long time
component deduced from visible-pump mid-IR-probe experiments [19] and ~3 from
12
time-resolved ps fluorescence studies [12]. The driving force for ESPT is significantly
weaker in pure water than in the acetate-water system, hence requiring more proton
tunneling through a higher reaction barrier. Therefore we observe the retardation of both
PA* decay and PB* rise on the 4.5–5 ps timescale in water, in comparison to the initial
1.6 ps PA* decay time constant in 2 M acetate water solution [27].
Caution is needed though when comparing these results because the spectral
source from which the time constants are derived is fundamentally different, and various
electronic and vibrational contributions might be involved. Our kinetic analysis is based
on the S1 vibrational peak intensity over a broad spectral range (instead of a broad
electronic response, or broad vibrational peaks over limited spectral range), which should
in principle be more accurate in capturing the structural snapshots of photoexcited
molecules in action. The observed KIE for the short time component of PA* decay (e.g.,
the 1048/1042 cm-1 mode in H2O/D2O) and PB* rise (e.g., the 1138/1136 cm-1 mode in
H2O/D2O) of HPTS is similar in magnitude to the ESPT process in solvated wtGFP,
wherein the observed C=N stretching mode redshift has a time constant of 5.1/22.0 ps for
the chromophore embedded in the protein pocket in H2O/D2O, respectively, so the KIE is
4.3 [9]. This similarity suggests that both the short and long time components of ESPT
from HPTS to water involve some significant and active proton transfer motions,
accompanied by the optimization of nearby heavy atoms likely within the ESPT chain on
the ps timescale, not merely the formation of contact pairs through highly specific proton
wires [18, 22]. The long time constant (>80 ps) sees diffusion playing a dominant role, as
PB* and the proton acceptor molecule are likely separated by 4 or more water molecules
[21], so ESPT and the charge transfer take much longer to complete. It is also notable that
these high-frequency vibrational modes mainly probe structural evolution after ESPT
efficiently proceeds following the initial preparation stage, wherein the impulsively
excited low-frequency modes emerge with various time constants (discussed below) on
the dynamic multidimensional PES and are sensitive to the instantaneous molecular
conformations of asymmetric HPTS on the fs to ps timescale.
Sequential emergence of various low-frequency modes gating ESPT. Following the
400 nm photoexcitation, we observe the multi-staged onset of PA* and PB* vibrational
modes. The optical excitation accesses the singly excited 1* state [10, 15, 19, 49], and
generally donates the photoexcitation energy to the totally symmetric Raman-active
vibrational modes that exhibit strong FC activities [19]. The sub-ps time constants
observed in our FSRS experiments on photoexcited HPTS in water range from 140–680
fs, a pre-ESPT timescale. The 952, 1048, and 1285 cm-1 modes rise with time constants
of 140, 210, and 190 fs, respectively, which strongly argues that these are readily
accessible PA* modes such as ring-HOOP motions, in-plane ring deformation, and ring
C–O(H) stretching motions. The prompt electronic redistribution over the aromatic ring
system upon photoexcitation induces these atomic motions, which have high Raman
polarizabilities along the FC slope and are strongly coupled to the electronic
redistribution process and exhibit excited-state vibrational modes at shifted positions
from the ground-state peaks. It is noteworthy that the emergence of various excited-state
vibrational modes particularly in the low-frequency region is intimately related to FC
dynamics of photoexcited HPTS as it undergoes conformational motions starting from
time zero. This ultrafast initial phase of excited-state structural evolution is likely
13
accompanied by intramolecular vibrational energy redistribution (IVR) processes [5, 43,
50-54], however, it is the relative equilibrium geometry displacement between S1 and
higher lying electronic excited states (or virtual state that is off-resonance) dictating the
FSRS peak intensity detected here by the Raman pump-probe pair. FSRS thus provides
the unique and laudable capability to monitor a multitude of vibrational modes
simultaneously and pinpoint the structural evolution pathway as ESPT initiates and
proceeds on the anharmonic S1 PES that is changing in real time. Certain vibrational
modes are promptly Raman active upon initial vibronic wavepacket generation on S1,
whereas some other vibrational modes become Raman active later in time because they
need a rather distorted molecular conformation to acquire significant Raman intensity.
Furthermore, the vibrational peak kinetics plot in FSRS is not limited by the vibrational
coherence decay typically on the few ps timescale because the time-delayed Raman pspump-fs-probe pair can generate multiple vibrational coherences due to the broad
bandwidth of the probe pulse to monitor the evolving molecular conformation along
multidimensional reaction coordinates. Therefore, the microscopic molecular snapshot
(i.e., HPTS conformational dynamics in S1 before fluorescence) is vividly available in
time-resolved excited-state FSRS. In particular, the observed transient vibrational modes
promptly following photoexcitation might be the dominant atomic motions contributing
to the proposed Stokes shift of photoexcited HPTS in H2O with the time constant of ~1 ps
[22], which is a separate and distinct phenomenon (i.e., preparation stage) from the
subsequent deprotonation process for PA*PB* population transfer (i.e., ESPT stage).
Regarding the low-frequency region, the most transient 191 cm-1 mode is
impulsively and coherently excited by the broadband photoexcitation pulse. It rises with a
~320 fs time constant and decays with a ~540 fs time constant. This in-plane ring skeletal
symmetric breathing motion has a large projection onto the initial reaction coordinate for
ESPT, coherently modulates the dihedral angle between the HPTS hydroxyl group and
the H-bonded water molecule (with a less optimal geometry in S0), and promotes some
immediate solvation process that accommodates the increased acidity of photoexcited
HPTS. The 540 fs decay component indicates that HPTS starts to break the original ring
coplanarity on that timescale, and should match the onset of various ring wagging, ringHOOPs, and asymmetric ring deformation motions of PA*. This is indeed the case: the
108, 321, and 362 cm-1 modes show the rise time constants of ~630, 650, and 650 fs,
respectively, and match well with the calculated frequencies at ~109, 311, and 355 cm-1
(Table 1). The 630 cm-1 mode in Fig. 3 likely involves the PA* ring in-plane and OOP
deformations, e.g., a combination of ring breathing and wagging motions, as confirmed
by DFT calculations at ~631 cm-1 (frequency scaling factor 0.96). The peak area plot
shows strong oscillations before 10 ps with a period of ~3 ps (data not shown, see below
the frequency quantum beat discussion for some modes below 250 cm-1). This mode rises
after 300 fs and mainly decays on the 90 ps timescale, and appears to be more prominent
in water than the acetate water solution previously studied [27]. The interwoven transient
vibrational peak kinetics suggests that the ESPT reaction coordinate on the excited-state
PES is multidimensional and anharmonic in nature to efficiently facilitate PT, and is
finely tuned by the molecular environment surrounding the photoacid in solution. On a
related note, the 191 cm-1 mode in Fig. 3 is weaker and decays faster in water than that in
2 M acetate water [27], suggesting that this intrinsically conserved coherent skeletal
motion of photoexcited HPTS takes on different dynamics upon photoexcitation in
14
response to an altered microenvironment for ESPT, and is thus an integral part of the
initial chemical reaction coordinate in the PES for the photoacidity of HPTS.
A small population of PB* is generated on the sub-ps timescale as well, evinced
by the fact that the characteristic PB* modes of 276 and 460 cm-1 exhibit the fast rise
time constants of 550 and 680 fs, respectively, although with distinct relative amplitudes
(85% vs. 26%, Table 1). This result indicates that the ring-HOOP motions at the phenolic
end of the nascent PB* state rise earlier and faster than the pure wags of the HPTS 4-ring
system. Some HOOP motions may facilitate the immediate solvation of PB*. In contrast,
the other PB* mode at 1138 cm-1 is fitted after 1 ps to show the biphasic rise time
constants of 5 and 78 ps. We cannot precisely comment on the sub-ps dynamics of this
mode due to complication from the overlapping ephemeral PA* feature (see Figs. 5 and
7), however, the two time constants directly report on two sequential stages for ESPT,
with the latter one dominating the accumulation of PB* due to the diffusion-assisted
distance-dependent ESPT. The electronic charge flow from the HPTS hydroxyl group
back to the aromatic ring system occurs strongly in the anion [3, 27, 55], likely after the

initial PT within the HPTS–O···H–OH2 complex to generate HPTS–O( )···H(+)–OH2, and
prepares for further PT between the nascent ion pair and beyond.
The aforementioned ~1 ps delay or preparation stage (Figs. 5 and 7) at 1138 cm-1
shows complex vibrational dynamics, likely associated with the Stokes shift of HPTS in
conjunction with solvation but not PT [22]. The ephemeral signal is ca. 2 cm-1 to the blue
side of the PB* mode (see Fig. 3, within the rightmost red box), and appears within our
instrument response time dictated by the cross correlation between the actinic pump and
Raman probe pulses (~140 fs). It then quickly decays away with a ~600 fs time constant,
similar to the transient 1180 cm-1 mode dynamics nearby. We speculate this transient
spectral feature at very early time being the PA* mode in the FC region way before the
PB* formation at barrier crossing, correlated with the disruption of the equilibrium
solvation shell possibly involving some immediate solvent motions around HPTS.
Therefore the sub-ps decay is indicative of the vibronic wavepacket moving out of the
initial FC region, toward the ESPT region on the PES. The PB* formation then becomes
the dominant process, and the peak kinetics tracks the ESPT progress as HPTS gradually
transfers its phenolic proton to nearby water molecules through H-bonding chains.
It is useful to accentuate that we observe prompt appearance of the ~191 cm-1
mode following photoexcitation, analogous to the 195 cm-1 mode previously observed in
the HPTS-acetate-water system [27], suggesting that this particular low-frequency mode
is characteristic of the HPTS in-plane ring symmetric breathing motion that modulates
the intermolecular O···O bending within the immediate solvation shell. In other words,
this conserved PA* mode samples the phase space that includes better H-bonding
geometry between the proton donor and neighboring acceptor, and might be a direct
consequence of the electronic redistribution that weakens the original HPTS–OH···OH2 Hbond in S0 [3]. This vibrational motion is likely ubiquitous for HPTS in water-based
solution and has a large projection onto the ESPT reaction coordinate. As a result, this
mode effectively receives the photoexcitation energy and helps the impulsively generated
vibronic wavepacket slide down the PES slope in the FC region. Once this process
occurs, the other coherent low-frequency modes associated with PA* (e.g., 108, 321, 362
cm-1, primarily ring wags and ring HOOPs) start to facilitate the proton motion through
the immediate H-bond to one acceptor water molecule on the typical hopping time of
15
~1.2 ps [21, 48, 56]. That is why we see a prominent decay component of all these ringwagging motions on the 1 ps timescale (Table 1), since the solvent rearrangement also
affects PA* vibrational motions, particularly those concerning H-bonding at the phenolic
hydroxyl end. An enlightening comparison can also be made to our previous results for
HPTS in 2 M acetate water solution: the 106 and 321 cm-1 modes observed therein both
rise on the ~630 fs timescale and exhibit the dominant decay time constant of ~1 ps [27],
with a relative exponential fitting amplitude of 80–85% that agrees very well with the
component percentage derived here in pure water. As more nearby water molecules start
to align along the ESPT chain connecting to the HPTS hydroxyl group, the ring wags
associated with the nascent charge-separated HPTS(–)–O···H(+)–OH2 at ~460 cm-1 report
on the formation of extended H-bonds with increased number of better-oriented water
molecules, presumably with a reduced coordination number in comparison to that in bulk
water [14, 24].
It is crucial to point out that photoexcited HPTS in pure water represents a
different case from the 2 M acetate water environment studied before [27]. In pure water,
there is no longer the strong donor-acceptor potential gradient between HPTS and acetate
to drive ESPT, and there are less effectively H-bonded or closer HPTS-acceptor pairs
already formed in S0. As a result, particularly after the vibrant molecular events (i.e.,
coherent skeletal motions) within the first 1 ps and some proton hopping to the immediate
H-bonded water molecule, a significantly greater amount of work is needed to bring more
nearby H2O molecules, which are over larger distances than those in the acetate water
case, into optimized H-bonding geometry with the nascent charge-separated HPTS(–)–
O···H(+)–[OH2]n=1,2... complex. That is why we observe a ~4.5 (24) ps PA* decay
component and a concomitant ~5 (21) ps PB* rise component in H2O (D2O), representing
further structural evolution of HPTS on S1 beyond the FC region.
The sub-ps rise time constant of 220 fs for the 1139 cm-1 PB* mode in the 2 M
acetate water system is absent in pure water, partially obscured by the overlapping PA*
mode that shows strong activity in the same spectral region up to 1 ps (Figs. 5 and 7).
This finding indicates that the absence of an effective proton acceptor such as the acetate
ion largely turns off direct ESPT over short distances through highly polarizable H-bonds
and much reduced proton transfer barrier on the <300 fs timescale. We suspect that a
small portion of the HPTS hydroxyl proton still manages to hop over to the immediately
adjacent H2O molecule that is originally H-bonded to the hydroxyl group of HPTS, but
only after some small-scale molecular reorientation of H2O occurs in ~1 ps to strengthen
that existing H-bond following photoexcitation. This agrees with the aforementioned subps emergence of various PA* low-frequency modes, and the accompanying solvent
motions within the first solvation shell. Except for the 276 cm-1 mode, a small percentage
of the PB* peak intensity rises on the timescale of ca. 1–20 ps, while the majority of the
PB* peak intensity rises on the timescale of ca. 80–200 ps in H2O and D2O (Tables 1 and
2). The ensemble-average approach of the FSRS measurement dictates that we observe
the overall time constants, with the clear distinction of two processes attributed to
establishing effective H-bonds with a few water molecules nearby, and to separating the
deprotonated HPTS (PB*) from the rest of the H-bonding chain as well as solvating the
nascent charge-separated molecules. The latter process is very likely to be diffusioncontrolled, but since the H-bonding chain is longer and more asymmetric [48] by then,
the deduced KIE of ~2.6 is smaller than the value (4.2) from the former PT process. The
16
majority of the PA* decay and PB* rise occur within that latter diffusion-controlled
process, meaning that ESPT can only effectively occur after the ~5 (21) ps heavy-atom
geometric optimization of the H (D)-bonding chain connecting the photoexcited HPTS
and the acceptor H2O (D2O) molecule via a few bridging H2O (D2O) molecules.
The time-resolved frequencies of the relatively sharp 172 and 215 cm-1 modes
(marked by vertical dashed lines but without labels in Fig. 3) after ~1 ps are compared
with the frequencies of the 108 and 125 cm-1 modes. The 125 cm-1 mode clearly shows a
delayed onset of ~1 ps (Fig. 8), whereas the 108 cm-1 mode rises promptly after
photoexcitation. It is interesting to find that the frequency quantum beats (data not
shown) displayed by these low-frequency modes (below 250 cm-1) are rather pronounced
on the 1–10 ps timescale, with a period of ~3 ps that corresponds to a modulation
vibrational mode at ~11 cm-1. The frequency oscillation of the 172 cm-1 mode is almost
180° out-of-phase to that of the 215 cm-1 mode. It is thus plausible to attribute the two
modes to PA* and PB*, respectively, assuming that the actively modulating 11 cm-1
vibrational mode survives the ESPT barrier crossing, and plays integral roles during both
the reactant (PA*) consumption and the product (PB*) generation. The frequency
oscillations of the 108 and 125 cm-1 modes are approximately in phase with each other,
commensurate with the mechanistic picture of sequentially observed PA* modes in
gating multi-staged ESPT. The peak intensities are expected to exhibit the quantum
beating effect as well, given that the nonlinear Raman polarizabilities of HPTS vary
during the low-frequency modulation period. This can be seen clearly in Fig. 9 wherein
the 952 cm-1 peak intensity is strongly modulated by a vibrational mode at ~93 cm-1
within 1 ps, suggesting that both modes are anharmonically coupled (likely mechanical
coupling) during the ESPT preparation stage [9, 39, 57]. Further experiments are planned
to unravel the low-frequency modes that remain active up to ~5 ps and anharmonically
couple to other low-frequency modes as seen here. More stable white-light probe pulse
and finer time delay steps are needed with improved SNR to reveal the quantum beats of
various vibrational modes particularly in the low-frequency region at early time, and to
provide deep insight on the multi-faceted PES of HPTS following photoexcitation.
Scheme 1. Multidimensional reaction coordinate and multi-staged ESPT from HPTS to
water: fs activation of skeletal modes gates the ps heterogeneous PT through optimizing
intervening water molecules
Multi-staged ESPT from HPTS to water with vivid conformational details. The fs
fluorescence up-conversion and pump-probe spectroscopies on HPTS in water have
revealed two ultrafast steps (300 fs and 2.5 ps) preceding the relatively slow diffusionrelated proton transfer step (87 ps) [3, 15]. The proposed mechanism involves solvation
dynamics, the formation of an ion contact pair, and the dissociation into free ions.
Solvent reorganizations play an important role to reduce the reaction barrier and to
accommodate the solvent-bridged ion contact pairs, and we have found previously that
the heterogeneity of this H-bonding network for ESPT affects the overall dynamics
observed for individual vibrational modes of the photoexcited HPTS in the presence of
acetate base [27]. The reported time dependence of the photoexcited PA* in water is
17
biphasic with decay time constants of ~3 ps (30%) and 100 ps (70%) from the pumpprobe measurements [18]. In another fs UV-pump IR-probe study on HPTS in water, the
transient absorbance for frequencies below 2850 cm-1 shows the multiexponential decay
with 300±200 fs, 3.0±1.5 ps, 90±30 ps, and 200±50 ps time components [19]. Our kinetic
analysis of the 1048 cm-1 PA* mode of HPTS in water reveals the bi-exponential decay
time constants of ca. 4.5 and 150 ps, which can be considered similar to previous results.
However, we believe that the values derived from the current time-resolved FSRS peak
kinetic analysis are more accurate since a number of vibrational marker bands are
spectrally separated from each other (Fig. 3), and can be analyzed simultaneously and
independently (Figs. 4–9). We also find that within the preparation stage for ESPT,
multiple low-frequency PA* modes coupled with some motions of the immediate Hbonding partner (Table 1) show pronounced activities on the 300 fs–1.2 ps timescale. In
addition, there is the subtle difference between the vibrational mode intensity observed
here and the concentration of the molecular species, and it is the interplay between
electronic and vibrational motions that leads to the polarizability change dictating Raman
peak intensity, not simply due to the concentration of the species involved. Therefore it is
more appropriate to discuss the non-equilibrium vibrational dynamics and KIE deduced
from FSRS data in the context of specific atomic motions of transient conformational
states, rather than a simple physical kinetic model that builds on equilibrium constants
and thermodynamics of various molecular species involved. In addition, the timeresolved FSRS peak intensity reported here effectively tracks the detailed structural
evolution starting from the FC region toward the product state on the excited-state PES,
because the Raman polarizability continues to change for vibrational normal modes as the
photoexcited molecule evolves on the multidimensional reaction surface with a complex
landscape and various equilibrium positions for participating vibrational modes.
The 1138 cm-1 PB* mode displays the biphasic rise with time constants of ~5 and
78 ps, which corroborates the short time component of PA* decay as the initial phase of
ESPT involving a few water molecules, although the predominant ESPT process for PB*
production occurs on the 78 ps timescale. This observation confirms the existence of
some initial stage that modifies the population of PA* but does not significantly generate
PB*. This could be due to some intermediate conformations of photoexcited HPTS that
have already broken the initial coplanarity of the aromatic ring system, have formed
contact pairs with neighboring proton acceptors reorienting on the ca. 5 ps timescale in
water, but have yet to conduct the long-range diffusion-controlled proton/charge transfer
and eventually separate the ion pairs. The relative large KIE observed in our FSRS data
confirms that after the preparation stage wherein the sequential observation of various
low-frequency modes is staple, heavy atom optimization is needed during the first phase
of ESPT on the few ps timescale, as the proton hops incoherently through more or less
randomly coiled water wires [25] that are asymmetric. The ~5 ps timescale is consistent
with the previously reported optimal ESPT distance of going across two water shells [1,
21, 25], where each water molecule donates two H-bonds over which the proton charge
gets transferred. The ~1.6 ps time constant previously observed for HPTS in 2 M acetate
water solution [27] can be understood in that the acetate ion effectively establishes a large
number of H-bonded ―reactive complexes‖, and reduces the initial acid-base distances to
sufficiently small values. This also explains the small KIE value of ~1.4 observed in
18
those tightly H-bonded systems involving the acetate ion as the adiabatic PT process
resembles the case for largely asymmetric, strongly downhill reactions.
The wealth of structural dynamics information provided by our time-resolved
excited-state FSRS spectra reveals that upon photoexcitation, the asymmetric HPTS
undergoes ESPT in water through multiple reaction stages: (1) the almost instantaneous
electronic redistribution leads to some swift in-plane ring deformation and ring-HOOP
motions on the 200 fs timescale (e.g., PA* S1 modes at 1048, 952 cm-1); (2) the facile inplane ring-breathing motion (e.g., 191 cm-1, PA*) then emerges with the 320 fs rise time
constant, modulating the relative geometry between the directly H-bonded HPTS
hydroxyl group and H2O molecule; (3) the ring wagging and phenolic HOOP motions
(e.g., 108, 321, 362 cm-1, PA*) subsequently gain Raman intensity with the 630–650 fs
rise time constant, in correlation with the diminishment of the in-plane ring breathing
motion with a 540 fs decay time constant, as well as the waning of the ring-HOOP
motion adjacent to the phenolic ring with a 600 fs decay time constant; (4) a small
amount of proton transfer starts to occur with the appearance of these PA* ring wagging
motions in S1, and characteristic ring wags associated with the deprotonated HPTS (PB*,
276 and 460 cm-1 modes) begin to accumulate on the 550–680 fs timescale; (5) The PA*
wagging motions start to diminish on the ca. 1 ps timescale, indicative of a solvation
process that mainly reorients the directly H-bonded H2O molecule to be in a more
favorable H-bonding geometry with HPTS [21, 27, 48], and the initial ground-state 4-ring
coplanarity is broken as the molecule reaches the charge-transfer state [15, 19, 49]; (6)
ESPT occurs through various-length H-bonding chains, accompanied by some tardy PA*
motions such as the 125 cm-1 mode, generating PB* with two distinct time constants of
~5 ps and 80 ps. The few ps component suggests that ESPT is a heterogeneous process
for HPTS in the labile H-bonded water matrix, since more H2O molecules nearby need to
be optimized to establish efficient H-bonds to transfer that phenolic proton. The observed
KIE of 3–4 is indicative of the weak H-bonding nature and possibly also longer Hbonding chains between HPTS and water, characteristic of the nonadiabatic PT where the
solvent fluctuations modulate both the PA* and PB* PES of the chromophore.
Besides the fundamental importance in elucidating the chemical reaction
mechanism of photoacidity, HPTS is also a fluorescent dye and pH sensor [58], and small
organic fluorophores have been powerful research tools to enable bioimaging with novel
insights into both cellular and molecular processes [59, 60]. This puts our work into
perspective for bioengineering and bioimaging. When the chromophore is part of a
protein sequence and strategically embedded in the protein pocket, wtGFP being the
perfect example for these genetically encodable biomarkers, the biofunctions can in
principle be illuminated step by step with unprecedented precision [9]. Whether or not
this precision extends into both the spatial and temporal regimes for practical bioimaging
is another question, and extensive work has been done in both areas to develop ultrahighresolution microscopy to overcome the diffraction limit [61], as well as to collect timeresolved cellular-level images in situ and in real time [62]. All these exciting applications
depend upon a photostable, bright, and controllable photosensitive reagent, which we can
understand deeply using the powerful toolset such as FSRS described here and then
perform targeted design at the molecular level to efficiently improve their
biofunctionality.
19
5. Conclusion
We have used the newly developed femtosecond stimulated Raman spectroscopy
(FSRS) to study the excited-state structural dynamics of HPTS in pure H2O and D2O
following 400 nm photoexcitation. The simultaneously high spectral and temporal
resolution of the apparatus enable the collection of time-resolved excited-state FSRS
spectra of the photoexcited chromophore as it transfers its phenolic proton to the labile
molecular water H-bonded network in real time. The non-equilibrium spectroscopic
approach reveals the multidimensional reaction coordinate on the excited-state PES for
intermolecular ESPT, wherein the sequentially emerged low-frequency skeletal motions
gate and/or facilitate ESPT in H2O on multiple timescales of 620±50 fs, ~4.5 ps, and
~100 ps. The observed KIE upon deuteration is in the vicinity of 3–4 for the latter two
time constants. We attribute the first sub-ps component to the preparation stage for ESPT,
which involves the decay of the transient 191 and 952 cm-1 PA* modes, and the rise of
the 108, 125, 276, 321, 362, and 460 cm-1 modes. The 276 and 460 cm-1 mode intensities
show the biphasic exponential rise, and are most likely associated with the ring wagging
and some HOOP motions of PB*. The 108, 321 and 362 cm-1 modes also show a
predominant (relative exponential fitting amplitude at 75–80%) decay time constant of
1.1 ps, matching the reorientation dynamics of a single water molecule possibly in direct
contact with the phenolic hydroxyl group of HPTS. The solvent rearrangement along the
H-bonding chain connecting HPTS and water molecules following photoexcitation plays
a dominant role in the ps dynamics of ESPT, and can be attributed to the nonadiabatic
proton transfer in comparison to the faster adiabatic ESPT when strong bases are present
in water solution. The observed ESPT on the 5–200 ps timescale in H2O/D2O involves
several water molecules being brought into better H-bonding geometry with the
photoexcited HPTS molecule, which later undergo diffusion-assisted ion-pair separation.
It is noteworthy to summarize the convincing evidences for a number of coherent
low-frequency vibrational modes to play the important functional gating role [4, 9, 27,
43, 63-65] in ESPT of HPTS in aqueous solution, besides the kinetic analysis of their
FSRS intensities. (1) They get impulsively excited pre-ESPT and exhibit different
dynamics. The deprotonated form PB* has the characteristic 1138 cm -1 mode that
redshifts from the 1154 cm-1 for PA at S0, and Fig. 7 shows that the 1138 cm-1 mode starts
to rise after 1 ps. The dwell between the observed low-frequency modes and ESPT is
small, indicative of causality. (2) These low-frequency modes significantly modulate the
intermolecular distance and/or geometry between HPTS and the neighboring H-bonding
acceptors, providing the appropriate atomic displacements to start optimizing H-bonding
chains and gate ESPT. (3) Quantum beats exist for a number of vibrational modes,
indicative of anharmonic coupling between various conformational motions of HPTS
capable of ESPT in water. (4) Certain conserved low-frequency modes exhibit different
dynamic behavior in response to different acceptor molecules. The key to retrieve the
underlying system Hamiltonian [9, 66, 67] is to observe mode-dependent vibrational
dynamics starting from time zero for HPTS in various external microenvironments, and
to simultaneously monitor a wide array of vibrational modes including the reactant,
intermediate and product with enough (i.e., fs) time resolution. We can then firmly
establish the causative connection between the observed coherent low-frequency modes
and ESPT via the temporal and structural correlation of the associated transient
20
molecular motions. FSRS thus renders an emerging powerful structural dynamics tool to
elucidate the choreography of ESPT from HPTS to water throughout the reaction, and
paves the way to study other photosensitive molecules with biological relevance on their
intrinsic reaction timescales in aqueous solution.
Acknowledgments
This paper is dedicated to Robin M. Hochstrasser who for over 50 years pioneered in
many fields of modern molecular spectroscopy and contributed deeply to our
understanding of the interplay between conformational dynamics and chemical reactions.
We thank the financial support from the Oregon State University Faculty Research
Startup Fund, and the College of Science Venture Fund Award to C. Fang. We are also
grateful to Yanli Wang and Longteng Tang for sample preparation, and to Breland Oscar
and reviewers for helpful discussion.
21
TABLE 1: Representative Vibrational Peaks of HPTS in H2O Observed in FSRS
FSRS peak
freq.a (cm-1)
cal. peak freq.b
(cm-1)
kinetics of the Raman
peak area
major
species
symbol
vibrational mode
assignment
PA*
HPTS 4-ring OOP wags
PA*
In-plane ring translation
with huge nearby water
translational motionc
PA*
Intermolecular O···O stretch
between the hydroxyl and
the acceptor
PA*
In-plane ring skeletal
breathing with
intermolecular O···O
bending at the phenolic ende
PB*
HPTS ring wags with the
phenolic COH HOOP
motions, and H-bonded
water HOOP motions
PA*
In-plane ring deformation
with some ring HOOPs
PA*
In-plane ring deformation
with significant COH
rocking motion with some
phenolic COH HOOPs
PB*
HPTS ring asymmetric
wagging motion
PA*
HPTS ring-H HOOPs and
in-plane ring deformation
PA, PB /
PA*
In-plane asymmetric ring
deformation with some
phenolic CO stretching
PB*
Phenolic CO···(H) rocking
and nearby ring-H rocking
(+) 630 fs
108
109
(–) 1.1 ps (75%);
1.7 ns (25%)
(+) 650 fs (55%);
125
132
5.3 ps (45%)
(–) 270 ps
143
150
Quantum beats with
~350 fs periodd
(+) 320 fs
191
208
(–) 540 fs
(+) 550 fs (85%)
276
271f
110 ps (15%)
(+) 650 fs
321
311
(–) 1.1 ps (80%);
75 ps (20%)
(+) 650 fs
362
355
(–) 1.2 ps (80%);
80 ps (20%)
(+) 680 fs (26%);
460
453f
130 ps (74%)
(+) 140 fs;
952
950
(–) 600 fs (78%)
90 ps (22%)
(+) 210 fs
1050 / 1048
1004g
(–) 4.5 ps (32%);
150 ps (68%)
(+) 5 ps (12%);
1138
1137f
78 ps (88%)
h
22
1154
1149i
N/A
PA
Phenolic COH rocking and
nearby ring-H rocking
PA*
Phenolic CO stretch and
strong ring-H & COH
rocking motions
(+) 190 fs
1285
1287
(–) 4.3 ps (40%);
215 ps (60%)
a
Observed Raman frequencies of the excited state as well as ground state FSRS peaks of
11 mM HPTS in pure water solution (pH≈6).
b
RB3LYP-DFT calculations are performed using 6-311G++(2d,2p) basis set for PAHPTS in aqueous solution in complex with a H-bonding water molecule at the phenolic
hydroxyl end. Calculation results with three H-bonding water molecules nearby show
slightly non-coplanar ring structure of HPTS in the ground state, and some OOP motions
mixed with the above-mentioned in-plane motions. Solvent effects are included by the
IEF-PCM-H2O model. The calculated vibrational frequencies are all scaled by a factor of
0.96.
c
This mode mainly involves the translational motion of the nearby H-bonded water
molecule at the phenolic hydroxyl end of HPTS. If the ring coplanarity is disrupted due to
the presence of more water molecules within the H-bonding distance, some slight OOP
motions are then mixed in. The main effect of this vibration is to significantly modulate
the intermolecular O···O distance between HPTS and the water molecule nearby.
d
The detailed kinetic plot is not attempted due to the strong oscillatory pattern and
ephemeral nature of the time-resolved peak integrated intensity (i.e., area) data. The
mode disappears within 1 ps. This mode involves some in-plane ring translational
motions and significant modulations between the ring hydroxyl and the neighboring
water molecule.
e
This ring skeletal breathing motion modulates the intermolecular (HPTS–)O–H···O(–
H2) angle and distance between the donor and H-bonded acceptor molecule in the same
ring plane. Slight OOP ring motions might be present if more water molecules are within
the H-bonding distance with the phenolic hydroxyl group, and modify the ground-state
geometry of HPTS in aqueous solution to some extent.
f
These PB* modes are approximated using the RB3LYP 6-31G+(d,p) calculation for PBHPTS in aqueous solution with a H-bonding water molecule at the phenolic hydroxyl end
(now with a C=O bond). In reality, the excited-state mode of PB* is in a partially
deprotonated configuration, which could significantly deviate from the simple calculation
of the corresponding ground-state mode in completely deprotonated PB.
g
This rather large discrepancy between the calculated and observed PA, PB mode is
interesting, as the DFT calculation correctly captures the trend of this mode being
unshifted from PA to PA···H2O, and from PB to PB···H2O. This mode seems to be
relatively insensitive to the electronic distribution over the ring system but deviates from
23
the realistic solution configuration. Given that it consists of large-scale in-plane ring
deformation and CO stretch at the phenolic hydroxyl end, it probably has cancellation
effect when electrons redistribute. This mode broadens in S1 compared with S0, and
shows characteristic rise-decay kinetics attributed to PA*, which has a ~2 cm-1 redshift to
the ground-state vibrational frequency of PA.
h
The double-exponential fit of the peak intensity kinetics plot shows a time-zero offset of
ca. 1 ps (Figs. 5 & 7). This is consistent with the delayed onset of this PB* vibrational
mode after the overlapping PA* mode disappears in the spectral region within ~1 ps (Fig.
3, within the rightmost red box), the preparation stage for ESPT.
i
Upon adding three water molecules within H-bonding distances to the phenolic
hydroxyl end of HPTS in the geometrically optimized Raman frequency calculation, the
DFT results show a slightly blueshifted mode at 1161 cm-1. Since the observed groundstate frequency is at ~1154 cm-1, it probably suggests that the first solvation shell of
HPTS at the phenolic end contains 1–2 H-bonded water molecules.
TABLE 2: Representative Vibrational Peaks of HPTS in D2O Observed in FSRS
FSRS peak freq.a
in D2O (cm-1)
kinetics of the
Raman peak area
major species
symbol
vibrational mode
assignment
PA*
In-plane asymmetric
ring deformation with
some phenolic CO
stretching
PB*
Phenolic CO···(D)
rocking and nearby
ring-H rocking
(+) 610 fs
1042
(–) 24 ps (40%);
650 ps (60%)
(+) 21 ps (12%)
1136
200 ps (88%) b
a
Observed Raman frequencies of two excited-state FSRS peaks of 11 mM HPTS in D2O
solution (pD≈6). Both peaks show some small frequency redshift to the corresponding
vibrational modes in H2O, probably due to the collective atomistic motions involved.
b
The double-exponential fit of the peak intensity kinetics plot shows a time-zero offset of
ca. 1 ps (Fig. 7). This is consistent with the aforementioned delayed onset of the PB*
vibrational mode at 1138 cm-1 in H2O.
24
Figure captions
Fig. 1. Schematic of the newly developed femtosecond stimulated Raman spectroscopy
(FSRS) in our laboratory [27]. The output laser beam from a Coherent femtosecond
regenerative amplifier is split to generate three beams: the actinic pump beam at 400 nm
(40 fs, 1 mW), Raman pump beam at 800 nm (3.5 ps, 6 mW), and Raman probe beam
with the wavelength range of 805–940 nm (30 fs, 100 nW). BS: beamsplitter, G:
reflective ruled diffraction grating (1200 grooves/mm, wavelength first order at 750 nm,
blaze angle θ=26.7°), CL: cylindrical lens, UM: pick-up mirror, ND: neutral density
filter, L: bi-convex lens (f=10 or 5 cm), SA: sapphire plate, LPF: long-wavelength pass
filter, PR1: fused silica prism pair, PR2: Suprasil-1 prism pair, P: polarizer, λ/2 WP: halfwavelength waveplate, and DL: delay line stage.
Fig. 2. Ground-state FSRS spectrum of HPTS in pure water (pH≈6). The two spectra
collected at two different spectrograph grating positions to expose the low-frequency and
high-frequency regions (10–2000 cm-1) are shown in red and black, respectively. The
chemical structures of HPTS in different H-bonding geometries with nearby H2O
molecules are depicted in equilibrium upon photoexcitation, transferring the proton from
its phenolic hydroxyl end to a proton acceptor H2O molecule, via intervening H2O
molecules. The two forms of HPTS, protonated (PA) and deprotonated (PB), are shown
in red and blue, respectively. The temporal evolution of PA* converting to PB* on the
excited state S1 can be found in the excited-state FSRS spectra in Fig. 3.
Fig. 3. Time-resolved excited-state FSRS spectra of 11 mM HPTS in water following ~1
mW 400 nm photoexcitation. The Raman pump is at 802 nm. The water-subtracted
ground-state FSRS spectrum of HPTS is scaled by 0.3 and plotted at the bottom for
comparison. The time delay up to 150 ps between the photoexcitation and Raman probe
pulses is noted beside each individual ground-state-subtracted excited-state spectrum,
with the vertical dashed lines marking the vibrational modes of interest from HPTS. Peak
frequencies in cm-1 are noted in the top portion of the figure: red for transient PA*
modes, black for other PA* modes, and blue for PB* modes. The red boxes enclose the
transient PA* modes that are predominantly active up to 1 ps. The blue box emphasizes
the low-frequency modes that rise after 1 ps. The magnitude of the stimulated Raman
peak strength is indicated by the double-arrowed vertical line in the middle of the figure.
Fig. 4. Time evolution of the 1048 cm-1 excited-state mode of 11 mM HPTS in water
following 400 nm electronic excitation up to 150 ps. The time-dependent stimulated
Raman peak intensity is fitted with a rising exponential (210 fs) and two decaying
exponential functions (~4.5 and 150 ps), convoluted with the ~140 fs instrument response
function determined from the cross correlation between the photoexcitation and Raman
probe pulses. The insert shows the very early time dynamics of the PA* mode up to 10
ps. The fits are shown in solid lines. The error bar represents one standard deviation from
the average value of multiple fitting procedures of the experimental data sets.
Fig. 5. Time evolution plots of three excited-state vibrational modes of HPTS in water
following 400 nm photoexcitation. The 1285 cm-1 (green), 1138 cm-1 (red), and 460 cm-1
25
(blue) modes can be attributed to PA*, PB*, and PB* modes, respectively (see text). The
fitting results are listed in Table 1. It is notable that due to the overlap of Franck-Condon
PA* modes and the nascent PB* mode around the ~1138 cm-1 spectral region, the doubleexponential fit of that vibrational mode is performed after ~1 ps (indicated by the vertical
dashed line).
Fig. 6. Time evolution of three low-frequency vibrational modes of photoexcited HPTS
in water following 400 nm electronic excitation. The stimulated Raman peak intensity is
taken as the integrated gain of the Raman peak with the solid lines showing the
multiexponential fits convoluted with instrument response time of 140 fs. The detailed
fitting results are listed in Table 1 for the 191, 276 and 321 cm-1 modes. It is evident (see
text) that the 191 and 321 cm-1 modes are two sequentially emerged PA* modes, while
the 276 cm-1 mode can be attributed to the nascent PB* state.
Fig. 7. Comparison between the peak intensity kinetic plots of the 1048/1042 cm-1 modes
in H2O/D2O (blue/red solid), and the 1138/1136 cm-1 modes in H2O/D2O (blue/red
dashed), respectively. The rise and decay time constants of HPTS in D2O are all
prolonged compared with those in H2O, shown as the disappearance of PA* and
appearance of PB* modes. Detailed fitting results regarding this isotope effect can be
found in Tables 1 and 2. The insert (expanded kinetics plot up to 10 ps) displays the PA*
preparation stage for ESPT in the HPTS-water system, before PB* starts to grow in
gradually after 1 ps. It is notable that following the preparation stage, the PB* mode
intensity rises faster in H2O than that in D2O.
Fig. 8. Temporal evolution of the peak intensities of the 108 and 125 cm-1 modes of
HPTS in water following 400 nm photoexcitation up to 150 ps. The 108 cm-1 mode rises
slower than the 1048 cm-1 mode in Fig. 4, and decays with time constants of ca. 1.1 ps
and 1.7 ns. The delayed onset of the 125 cm-1 mode is conspicuous from the figure,
which is also highlighted by the leftmost blue box in Fig. 3. The decaying behavior on the
ps timescale suggests that both modes are associated with PA*, but with different
mechanism to facilitate multi-staged ESPT from HPTS to water.
Fig. 9. Time-resolved peak intensity plot of the 952 cm-1 mode of 400 nm-photoexcited
HPTS in water. Pronounced intensity oscillations show up before 1 ps with a period of
~360 fs (similar to the kinetic plot of the 143 cm-1 mode intensity), which corresponds to
a modulating low-frequency mode at ~93 cm-1. The observed 108 cm-1 mode primarily
involves 4-ring OOP wags (Table 1) and has an closely matching frequency, hence could
be the modulating source via mechanical coupling. The structural origin for the proposed
anharmonic coupling between these vibrational modes in S1 along the ESPT reaction
coordinate is described in the text.
26
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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Scheme in Discussion Text
Graphical Abstract TOC Figure
A number of low – frequency modes are sequentially observed in photoexcited HPTS.
Evidence of coherent quantum beat in several low – frequency modes with anharmonic coupling.
The most transient low – frequency mode is the symmetric ring breathing of HPTS.
Excited – state proton transfer occurs nonadiabatically on hte 5-200ps timescale.
Kinetic isotope effect is 3-4 fot the two – stage ESPT components in water.