Time-Resolved Resonance Raman Spectroscopy: Exploring Reactive Intermediates

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SANGRAM KESHARI SAHOO AND SIVA UMAPATHY DEPARTMENT OF INORGANIC AND PHYSICAL CHEMISTRY INDIAN INSTITUTE OF SCIENCE BANGALORE -560012, INDIA

ANTHONY W. PARKER CENTRAL LASER FACILITY RESEARCH COMPLEX AT HARWELL SCIENCE &TECHNOLOGY FACILITIES COUNCIL RUTHERFORD APPLETON LABORATORY HARWELL OXFORD DIDCOT OXFORDSHIRE, UK OX11 0QX Time-Resolved Resonance Raman Spectroscopy: Exploring Reactive Intermediates

The study of reaction mechanisms involves technique. The simultaneous advances in con- efficient spectrometers, and high speed, highly systematic investigations of the correlation temporary time-resolved Raman spectroscopic sensitive multichannel detectors able to collect between structure, reactivity, and time. The techniques and computational methods have a complete spectrum. This review article will challenge is to be able to observe the chemical done much towards visualizing molecular provide a brief chronological development of changes undergone by reactants as they change fingerprint snapshots of the reactive interme- the experimental setup and demonstrate how into products via one or several intermediates diates in the microsecond to femtosecond time experimentalists have conquered numerous such as electronic excited states (singlet and domain. Raman spectroscopy and its sensitive challenges to obtain background-free (remov- triplet), radicals, radical ions, carbocations, counterpart resonance Raman spectroscopy ing fluorescence), intense, and highly spectrally resolved Raman spectra in the nanosecond to carbanions, carbenes, nitrenes, nitrinium ions, have been well proven as means for determin- microsecond (ns–ls) and picosecond (ps) time etc. The vast array of intermediates and ing molecular structure, chemical bonding, domains and, perhaps surprisingly, laid the timescales means there is no single ‘‘do-it-all’’ reactivity, and dynamics of short-lived intermediates in solution phase and are advanta- foundations for new techniques such as spatial- geous in comparison to commonly used time- ly offset Raman spectroscopy. Received 7 July 2011; accepted 25 July 2011. resolved absorption and emission spectroscopy. Index Headings: Time-resolved resonance Ra- * Authors to whom correspondence should be sent. E-mail: [email protected]; a.w. Today time-resolved Raman spectroscopy is a man spectroscopy; TR3 spectroscopy; Qui- [email protected]. mature technique; its development owes much nones; Charge transfer complex; Photobiology; DOI: 10.1366/11-06406 to the advent of pulsed tunable lasers, highly Isomerization; Inorganic complexes.

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INTRODUCTION studying biomolecular dynamics. How- fluorescence spectra occurs, the more ever, recently the time-resolved struc- intense fluorescence obscures the weak- he chemical mechanism of a tural elucidation techniques, such as er Raman features. This is particularly single reaction or a set of similar time-resolved Raman and infrared spec- so when working on biological mole- reactions is first hypothesized T troscopies, have provided dynamical cules in the visible region; however, and then predicted by a patterned information from a few femtoseconds moving into the ultraviolet (UV) or deep analysis of the reactants used and the final products formed. Due to the lack of to microseconds and days. The first UV region can avoid such problems complete experimental evidence, this experimental study of inelastically scat- whilst still benefitting from resonance tered light was reported by Raman and enhancement. The enhanced RR bands procedure can overlook the details of 7 the intermediates formed during the Krishnan in 1928. The contemporary are sensitive to structural and environ- reaction. Chemistry textbooks are full technique at that time received world- mental change and are selective in of descriptions of proposed mechanisms wide attention, though it had limited nature, which is essential for probing (albeit based on rational basic principles application due to technical limitations. different sites of a multichromophoric of chemistry) leading to the key inter- The invention of lasers and multichannel chemical/biological system, for example detectors in the 1960s and 1970s, macromolecular systems such as por- mediates formed within simple as well 9–11 as complex reactions, yet frequently respectively, led the renaissance in phyrins and proteins, independently. concrete structural evidence is lacking Raman spectroscopy. Since then we Thus, the first time-resolved reso- to support the hypotheses put forward. It have witnessed an explosion in Raman nance Raman (TR3) experiment probed is the responsibility of time-resolved modernization. Continuous wave (CW) the molecular structure of the reactive spectroscopists to provide experimental and short-pulse lasers, sophisticated intermediates generated from a 30 ns data that can be used to verify these spectrometers, charge coupled devices pulse radiolytic pump source. The work hypotheses. A recent example of such a (CCD), intensified CCDs, electron mul- was reported in 1976 by Weisberg and 12 case is the study of SN2 reaction tiplying CCDs, streak cameras, Rayleigh co-worker. In the early 1980s nano- - filters, and fluorescence rejection tech- second and picosecond (ps) TR3 exper- involving Cl and CH3I. The textbook and the research papers agree with the niques have provided today’s multiple iments were demonstrated using single 13–15 formation of a five-coordinated transi- approaches to Raman spectroscopy that and double laser pulses. With the tion state during one-step nucleophilic are applicable to all sample phases under technology developed across the nano- substitution. However, a recent finding any condition. Raman spectroscopy, second and picosecond time domain, using crossed molecular beam velocity which can be used to understand the TR3 has since been successfully applied map imaging suggests a roundabout molecular structure and the subtle to probe short-lived reactive intermedi- 1 mechanism involving CH3 rotation. changes in the bond length and bond ates produced in numerous chemical and 16–20 A typical photochemical reaction in angles during a reaction, is now consid- biological systems. Figure 1 pre- solution phase involves the evolution ered the ‘‘must have’’ technique in sents schematically the theme of RR, TR and decay of various short-lived reactive academia and industry.8 spectroscopy, and regions of TR3 spec- intermediates that must navigate through If lasers led to the renaissance in troscopy in the electromagnetic spec- a series of parallel competitive pathways Raman spectroscopy, it was short-pulsed trum and their application for important to produce products. Depending on the and tunable lasers that revolutionized it! photo-processes studied in different time detail required and timescales involved, Further, the resonance Raman (RR) domains. the experimentalist has an array of time- condition means the intensity of inher- In this article we focus on the detailed resolved (TR) techniques with which to ently weak Raman bands is selectively study of structure, reactivity, and dy- probe the reaction. Any time-resolved enhanced, up to 106 times, by tuning the namics of photogenerated intermediates technique uses an activation procedure excitation line into an allowed electronic probed by time-resolved resonance Ra- to initialize (pump) a change (reaction) transition. RR scattering occurs through man (TR3) spectroscopy in the nano- and the spectra of the intermediates are coupling of electronic and vibrational second to picosecond time domain. recorded using a suitable probe source at transitions. The Raman spectral profile Time-resolved absorption (TRA) and variable delay times with respect to the obtained under the resonance condition fluorescence (TRF) on the fast and pump. Apart from photolysis, pulse is different in comparison to the non- ultrafast scale are the oldest techniques radiolysis,2–4 thermal, chemical, and resonant Raman condition. In addition, and the most commonly applied for electrochemical methods have also been because of the resonance enhancement, studying kinetics of photochemical re- used to activate change. The chemically detection of components in a very dilute actions.21–23 TR3 spectroscopy has a initiated reactions typically involve rap- solution is possible. RR spectroscopy is major advantage over the UV-Visible id mixing, stopped-flow methods,5 or used primarily in the visible wavelength transient absorption method because it oxidation and reduction6 to form inter- region; however, the UV region is can selectively probe multiple interme- mediates. Though mixing methods only important for studying biomolecules diates with greater accuracy as well as allow time resolution in the microsecond and highly conjugated chemical sys- provide bond-specific structural details to millisecond (ls–ms) domain, tems. In certain spectral regions the of the intermediate. The structural stopped-flow remains, even in today’s resonance Raman laser causes fluores- changes and the kinetics (reactivity) ultrafast world, an excellent tool for cence. When overlap of Raman and information can be obtained by analyz-

1088 Volume 65, Number 10, 2011 ing the band shift and intensity of RR bands, respectively. The RR bands are also highly sensitive to changes in the substrate structure and the environment, which helps in studying microscopic molecular interactions. Of course, other time-resolved Raman methods are being employed and the reader is referred to various recent reviews describing femtosecond stimulated Raman spectroscopy (FSRS)24,25 and coherent anti-Stokes Raman spectroscopy (CARS).26 In the solution phase, each chemical modification means that the intermediate must equilibrate with the surrounding solvent molecules. Consider, for example, the simplest chemical reaction of all, a charge-transfer reaction between two molecules. As the electron transfer occurs between a donor and an acceptor, the nascent radical cation and radical anion can only dissociate and become solvated ions if the solvation energy is sufficient to overcome the columbic attraction between the positively and negatively charged intermediates. In effect, the separation step is equivalent to the two molecular systems having to ‘‘redissolve,’’ and in a polar solvent, ion solvation will be sufficiently encouraged (imagine dissolving NaCl in water) and occur at a rate fast enough to prevent back-electron transfer and the ions returning to the original reactants. On the other hand, in a non-polar solvent no ion stabilization occurs and the solvation is prevented and the reaction does not go to completion. An early success of TR3 was to follow the electron transfer process between anthraquinone and 1,2,4-trimethoxybenzene as shown in Scheme 1. This work reported the geminate ion pair formed within the reaction of triplet anthraquinone (AQ) and 1,2,4-trimethoxybenzene (TMB) in a medium polarity solvent (1,1,2,2-tetrachloroethane, TCE) and showed that the ions within the ion pair are not solvent separated.27

FIG.1. (A) Schematic energy level diagram for resonance Raman (RR) spectroscopy. (B) Scheme for time-resolved (TR) spectroscopies including possible short- lived reactive intermediates. (C) Time scale of various important photo processes studied by time-resolved resonance Raman (TR3) spectroscopy.

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electronic excitation involved is non- zero, i.e., it should be an ‘‘allowed’’ transition. Secondly, the Franck–Con- don factors (FC), hnjvihvjmi, should be non-zero. Four possible scenarios may be identified, which have been depicted in Fig. 3. If the potential energy surfaces (PES) of the ground and excited states have the same force constants (curvature) and the minima are not displaced SCHEME 1. Pathway for an electron transfer reaction between a triplet electron acceptor with respect to each other, the vibra- (A) and ground-state electron donor (D). Key: intersystem crossing (isc), diffusion (diff), tional eigen functions for both surfaces forward electron transfer (fet), dissociation (diss), hyperfine coupling (hfc), separation (sep), will be the same, (the case (A) in the recombination (rec), and back electron transfer (bet).27 [Redrawn with permission from E. Vauthey, A. W. Parker, B. Nohova, and D. Phillips, J. Am. Chem. Soc. 116, 9182 (1994). Fig. 3). Therefore, the Franck–Condon Copyright 1994 American Chemical Society.] factors will be zero. If the force constants are the same but the minima of the excited state PES is displaced The center-to-center interionic dis- tronic and vibrational transitions are with respect to that of the ground state, tance is 7.5 A˚ and the activation barrier coupled and the ground and excited the FC factors will be non-zero (case for ion separation is 0.04 eV. This electronic states can better be described (B)). Case (C) occurs when the force corresponds to the amount of energy as vibronic states. We deliberately skip constant of the excited state is different required to overcome electrostatic stabi- all the mathematical aspects describing from that of the ground state, so that the lization and increase the distance be- the resonance Raman effect, which are FC factors will be non-zero even if the tween the ion pair to 9.5 A˚ , allowing well documented in the literature.42–45 minima of the PES coincide. Finally, the solvent molecules to penetrate between In the case of a resonantly excited most general case (case (D)), occurs the ions. These results were obtained by transition from initial ground electronic when the excited state is both displaced monitoring the band shape changes of state jii to excited electronic state jfi, and has a different curvature with the ~1600 cm-1 resonance Raman band implementing the sum-over-state ap- respect to the ground state. þ 46 in the spectrum of TMB . As shown in proach, the polarizability tensor [aqr]if The B-term accounts for the vibronic Fig. 2, this band could be reproduced by is the result of four contributions coupling between the resonant excited fitting two Gaussian bands, the intensi- referred to as A, B, C, and D terms. Of state and another close lying electronic ties of which varied with time delay of these, the A and B terms are given state, with appropriate symmetry. The the pump and probe laser. Analysis of by42,43 the following expressions: magnitude of the coupling depends on the kinetics of this process at different X the coupling integral, hes, the product of 1 0 0 hinjv hivjm temperatures produced an Arrhenius plot A ¼ MqMr ð1aÞ vibrational overlap integrals, as well as which gave the thermodynamic infor- hc v -e--0 þ iC on the transition dipole moments of the mation. This example demonstrates di- ground state to each of these excited rectly how solvent effects can be X k states. Therefore, for the B-term en- 1 0 0 hse monitored by carefully selecting the B ¼ MqMr hancement of the bands, it is necessary h2c2 D- appropriate laser wavelength to tune s6¼e se that the electronic state that couples to into a specific intermediate. For the X the resonant state also has a non-zero hinjQkjv hivjm 1 above example the chosen laser wave- þ transition dipole moment. - -- þ iC h2c2 lengths were 540 nm and 460 nm to v e 0 - interrogate the AQ and TMB þ, re- X hk INSTRUMENTATION M0M0 es spectively. Therefore, an essential need q r D- DEVELOPMENT was to perform precursor time-resolved s6¼e es absorption (TRA) studies to identify the X Raman scattering is inherently weak hinjv hivjQkjm in nature and suffers from highly intense resonance Raman wavelengths required ð1bÞ -e--0 þ iC Rayleigh scattering and fluorescence. prior to TR3 work. The technical and v Intense CW and pulsed laser sources scientific growth using TR3 as a tech- where m and n are the vibrational have helped in decreasing the spectral nique has been reported from time to j i j i eigenstates of the ground electronic acquisition time, increasing spectral time in past few decades.28–41 state, jvi are the vibrational eigenstates resolution, and providing a wider Ra- THEORY OF RESONANCE of the excited electronic state of the man operational window spreading from RAMAN SCATTERING scattering species, and Mq is the transi- the deep UV (DUV) through the visible tion dipole moment. to the near infrared (NIR). In this section The electronic transition in a molec- From the expression for the A term, it development of instrumentation for four ular system is generally accompanied by is clear that this term will be non-zero if independent types of TR3 spectroscopy vibrational energy changes. The elec- the transition dipole moment of the operating in different time domains

1090 Volume 65, Number 10, 2011 along with the modalities for fluorescence rejection will be discussed. The techniques include single-pulse transient Raman, nanosecond TR3 spectroscopy, picosecond TR3 spectroscopy, and temperature-jump (T-jump) Raman spectroscopy. Single-Pulse Transient Raman Spectroscopy: A Reliable Tool in the Early Days. When employing high- power pulsed laser sources for recording Raman spectra, the molecular system should not be perturbed. Using pulsed excitation, the fraction of molecules getting photolyzed is represented by the photoalteration parameter F. The reader is referred to several reports in the literature describing numerical calcula- tion of F and its dependence on various parameters to choose laser power and solution flow rate for effective generation of reactive intermediates.47,48 The value of F for the pump pulse must be »1 for effective photolysis to generate large concentration of intermediates and that of the probe pulse must be kept at ,0.2. Time-resolved resonance Raman experiments may be carried out using both single and two-color laser pulses either in the nanosecond or picosecond time domain to investigate the photogenerated transient species. In a typical single-pulse experiment, the same laser pumps the sample to generate intermediates as well as probing them within the duration of its pulse width or through dividing the same beam to follow two optical paths, providing temporal separation. Raman spectra are recorded sequentially at low (concentration of intermediate is low) and high incident laser power (appreciably detectable concentration of intermediates) and the difference spectrum contains the time- resolved molecular fingerprint of the unknown intermediates. As a two-photon process, the intensity of Raman signals of the transient species when FIG.2. (A) Time-resolved resonance Raman study of the rate of separation of a geminate using a single laser increase linearly ion pair into free ions in a medium polarity solvent, pumping at 351 nm and probing at 460 with the square of the laser power. nm a solution of anthraquinone (electron acceptor) and 1,2,4-trimethoxybenzene (electron donor) in 1,1,2,2-tetrachloroethane at -5 8C. (B) Fitting of the ~1600 cm-1 band and its time Figure 4 shows the different TR3 evolution of the band shape as the geminate ion pair (red) and free in the solvent (green).27 spectral regions of bacteriorhodopsin [Reprinted with permission from E. Vauthey, A. W. Parker, B. Nohova, and D. Phillips, J. Am. (bR570) at low (Figs. 4A and 4D) and Chem. Soc. 116, 9182 (1994). Copyright 1994 American Chemical Society.] high (Figs. 4B and 4E) laser power range.47 At low laser power (F = 0.15) the Raman spectrum (Fig. 4A) is dominated

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artifacts a single laser can be used, either split into two beams or pulsed rapidly. This gives some limited control between the pump and probe time difference. Optical delays can provide difficulties in controlling the pointing of laser beams over large distances because of small off-axis movements to reflective optics as they traverse the delay lines. This means delays . 10 ns becomes difficult for small diameter beams with poor divergence, although modern adaptive optics that can be programmed to automatically adjust and realign beams may provide some relief. The two-color, two-pulse Raman experimental modality must be considered to be a remarkable development to obtain the real-time structural geometry and kinetics parameters of intermediates involving various dynamical processes. A typical experiment uses two pulse- synchronized laser sources. The pump pulse initializes the reaction, usually via electronically (photo) exciting the molecular system into higher excited states and the probe pulse interrogates the cascade of intermediates produced at variable delay times with respect to the pump. The Raman detection window is dictated by the probe wavelength. When the two different laser wavelengths are used, combining these laser beams at the sample is usually performed using beam splitters and/or dichroic mirrors, which permit transmission of one wavelength FIG.3. Schematic illustration of the four possible cases in A-term resonance Raman 43 and reflect another, providing a means to scattering with xg = xe in cases (A) and (C) and xg 6¼ xe in cases (B) and (D). [Reproduced from R. J. H. Clark and T. J. Dines, Angew. Chem. Int. Ed. 25, 131 (1986). Copyright 1986 make the two beams co-linear. The John Wiley & Sons, Copyright Wiley-VCH Verlag GmbH & Co. KGaA.] advantages of two-color two-pulse Ra- man experiments are (1) the time delay by the ethylenic stretching band (str.) of intermediate at 1518 cm-1 indicating between the pulses can be accurately bR observed at 1530 cm-1,which structural changes for the intermediate. varied and (2) the pump and probe corresponds to an unphotolyzed The single-color Raman difference wavelength can be optimized to fulfill ground-state signature. The spectrum spectrum is also known as transient the resonance condition. The picosecond recorded at high laser power (F = 1.9, Raman spectroscopy because of the and nanosecond TR3 spectra of inter- Fig. 4B) represents the contributions limited time resolution. The single-pulse mediates provide information from pi- coseconds to several nanoseconds and from both bR570 and photolytically method has been applied to a variety of generated intermediate J625. Again the molecules including organic, metal-cen- several nanoseconds to microsecond appearance of Raman bands in the tered, and biological systems.40 Though time scale, respectively. The delay region of 1174 cm-1 to 1203 cm-1 the single-pulse experiment has several between pump and probe pulse for provides information about the initiation advantages (cost effectiveness and ease obtaining nanosecond and picosecond of all-trans to 13-cis retinal isomeriza- of setup), it suffers from lack of accurate TR3 spectra is adjusted electronically tion. The above results overall indicate time resolution and the fact that very and optically, respectively. The combi- the initiation of the bR photocycle at high peak powers must be avoided as nation of both picosecond and nanosec- high laser power domain. The difference they can lead to artifacts stemming from ond provides the structural and spectrum (4C = 4B - 4A) clearly shows thermal heating, nonlinear interaction, dynamical information in picosecond to the downshifted Raman band of the J625 and sample degradation. To avoid such microsecond time scales.

1092 Volume 65, Number 10, 2011 FIG.4. Traces A and D correspond to low laser power (F = 0.15) unphotolyzed ground-state Raman spectra of bR570. Traces B and E correspond to the Raman spectra recorded at high laser power (F = 1.9) having information about both bR570 and intermediate J625. Traces C and F show the difference spectrum containing information about the structure of the intermediate state.47 [Reproduced from R. Van den Berg, D. -J. Jang, H. C. Bitting, and M. A. El-Sayed, Biophys. J. 58, 135 (1990). Copyright 1990 Elsevier.]

Nanosecond Time-Resolved Reso- laser should be chosen in such a way second, third, and fourth harmonics of nance Raman Spectroscopy. A typical that it must match with one of the the Nd:YAG laser (532 nm, 355 nm, and two-pulse experimental setup for per- ground-state electronic absorption pro- 266 nm, respectively), excimer laser forming nanosecond TR3 spectroscopy ﬁles. This is required for effective (308 nm), nitrogen laser (337 nm), and is schematically depicted in Fig. 5. The pumping and generation of intermedi- frequency-doubled dye lasers having 5– wavelength of the nanosecond pump ates in detectable concentration. The 10 ns pulse width and 10–100 Hz

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FIG.5. A Schematic representation of possible components of ns TR3 setup. OPO: Optical Parametric Oscillator, RS: Raman Shifter, DG: Delay Generator, PB: Pellin-Broca Prism, DM: Dichroic Mirror, PD: Photodiode, L1: Focusing Lens, L2: Collection Optics, NF: Notch Filter (required for single monochromator), MONO: Double Monochromator (single and triple monochromator can also be used), CCD: Charge Coupled Device, ICCD: Intensified CCD, EMCCD: Electron Multiplying CCD, PDA: Photodiode Array, OSC: Oscilloscope, CP: Circulating Pump, and PC: Personal Computer. repetition rate are a few of the work- probe laser can be electronically varied experiments the sample must be flowed horses used as pump source in nanosec- by using pulse-delay generators to sufficiently fast to allow fresh sample to ond TR3 experiments. A number of trigger both flash lamp and Q-switch of be exposed to each laser pulse to avoid wavelengths can be produced for pump both lasers. A small portion of the pump the buildup of photoproduct and sample and probe by using the harmonics of and probe pulses is reflected and direct- damage. Sometimes the sample is Nd:YAG for pumping dye lasers,49,50 ed to a fast photodiode, which may be flowed through a free jet to avoid optical parametric oscillators (OPO),51 viewed on an oscilloscope to monitor unwanted scattering from the walls of and gas-52 and solid-state Raman shift- the pump–probe time delay. The liquid the capillary. ers.53 The output wavelengths from sample is continuously flowed through a Both the pump and probe pulses are these sources can further be manipulated quartz capillary or spinning NMR spatially and temporally overlapped on (doubling, tripling, mixing) to produce tube.54,55 Both serve to maintain deox- the sample capillary and loosely fo- various continuously tunable discrete ygenated samples, needed for triplet cused. The incident light after passing colors of monochromatic light spanning excited-state studies, as well as protect- through the capillary is collected at 908 from the DUV to the NIR region. The ing the experimentalist from toxic or 1808 by a collection lens and focused relative time delay between pump and chemicals. In pulsed laser repetition rate into the entrance slit of the monochro-

1094 Volume 65, Number 10, 2011 mator. The 1808 geometry is advanta- In common practice, the bandwidth of ed (frequency doubled or tripled) to geous as it helps to minimize light the laser should not exceed the spectral produce tunable output in the range of absorption by the sample, inner filter bandwidth of the Raman bands of 375–320 nm and 250–280 nm that can effects, and better collection of weak interest. It is appropriate to note here be used as the pump/probe beams. The scattered light. The spectrograph can be that the spectral resolution in ultrafast 800 nm output from the amplifier can be a single, double, or triple grating system experiments is obviously poorer than for frequency doubled to produce 400 nm depending on the stray light rejection nanosecond time-scale experiments. suitable for pumping one or two optical and light throughput required. The Further, because of the spectral width parametric amplifiers (OPA) and gener- multiple grating systems are better for of short pulses (,1 ps), conventional ating a continuously tunable wavelength stray light rejection, particularly at very two-pulse methods using femtosecond range in the visible (470-740 nm) and low wavenumbers, but results in lower pulses can’t be applied to extend the NIR (900-2700 nm) regions. Again the light throughput (maximum signal loss time resolution of time-resolved Raman OPA output can be mixed with the due to more optical surfaces). The spectroscopy to the femtosecond (fs) residual fundamental output from the gratings in the spectrometer disperse time domain. A newer set of nonlinear Ti:Sapphire amplifier to produce more the scattered light usually onto a cooled techniques called femtosecond stimulat- wavelengths in the UV and visible liquid nitrogen (or Peltier) charge-cou- ed Raman spectroscopy (FSRS)24,56 and regions. By a careful choice of wave- pled device (LNCCD), intensified CCD ultrafast Raman loss spectroscopy length manipulation the complete oscil- (ICCD), electron multiplying CCD (URLS)57–60 have been developed to lator–amplifier–OPA system can (EMCCD), or a photodiode array, which extend the time resolution into the provide working wavelengths (pump/ converts the light signal to an electrical femtosecond domain but are outside probe) for ultrafast TR3 experiments in signal read and displayed by a computer the scope of the current discussion. the range of 200–1100 nm with ample as the final Raman spectrum. Typical These days it is common to use energy for each excitation line. exposure times are in the 200 to 300 s picosecond pulses with a pulse width Quality control of the picosecond range with an average of two to three between 1 and 3 ps for ultrafast time- pulses utilizes autocorrelator systems. separate accumulations. Three separate resolved spectroscopy where the spectral The time zero, temporal overlap be- spectral measurements are carried out, width is about equal to the Raman band tween pump and probe laser fields, can (A) a pump-only spectrum, required if widths in solution phase, typically 12 befoundbyperformingatransient background signals occur, e.g., to obtain wavenumbers.61 absorption study of a ‘‘standard sample’’ a fluorescence profile, (B) the probe only Many methods are available for that matches the wavelengths being used (gives normal (ground) state Raman, and generating ultrafast pulses to perform and monitoring the change in intensity (C) the pump–probe spectrum, for each ps TR3 experiments.61–63 However, of the probe pulse as the nascent state is time delay. The known solvent Raman with the advent of solid-state technolo- produced by the pump or through an bands may be used for wavenumber gy, Ti:Sapphire oscillator/amplifier sys- optical Kerr effect experiment. The calibration. The final RR spectrum of the tems having greater pulse stability are relative time delay between pump and intermediate for a single delay time can popularly used for the above experi- probe source is varied through a com- be obtained by subtracting the probe ments. Figure 6 represents a schematic puter-controlled motorized translation only and pump only spectrum from the diagram of the basic components of a stage mounted with a retroreflector, pump–probe spectrum. Similarly, spec- typical two-pulse ps TR3 experimental which permits semi-automated signal tra at several delay times should be setup based on a Ti:Sapphire oscillator/ accumulation. Usually the probe beam recorded for kinetics analysis. amplifier system. is allowed to pass through the transla- Ultrafast (Picosecond) Time-Re- The fs Ti:Sapphire oscillator is tional stage (1 ft/30 cm path difference solved Resonance Raman Spectrosco- pumped by the 532 nm CW output from = 1 ns time delay). Both the pump and py. Nanosecond TR3 experiments have a diode-pumped Nd:YVO4 laser to probe beam are focused to 100–200 lm limited time-resolution (up to a few produce a tunable output of 720–850 diameter at the sample, which is flowed nanoseconds). To perform picosecond nm with 86 MHz repetition rate and through a 500 lm diameter open jet or a TR3 spectroscopy several factors need energy of 5–15 nJ/pulse. The oscillator capillary tube. The scattered light is to be carefully considered. Applying the output is used as a seed for a ps collected by a collection lens, dispersed Heisenberg uncertainty principle to an Ti:Sapphire regenerative amplifier, in a spectrometer, and detected by ultrafast transform limited Gaussian which is pumped by 527 nm output multichannel CCD detectors. Rayleigh pulse: from a CW diode-pumped Nd:YLF rejection filters are used as necessary Dx Dt » 2p ð2Þ intra-cavity doubled 1 kHz Q-switched when a single-stage spectrograph is laser. The output from the picosecond used. The data collection and analysis where Dx is the spectral width and Dt is regenerative amplifier generally operates in ps TR3 experiments are performed in the temporal pulse width. Therefore, Dk in the range of 750–840 nm with a 1 the same way as ns experiments, 2 -1 » k0/(cDt), Dk » 21 nm (324 cm ) for kHz repetition rate, 1 ps pulse width, although for ease of operation the 100 fs pulses with k0 = 800 nm. Thus, and average energy of 1–2 mJ/pulse. pump-only and probe-only approaches for a 2 ps pulse, a bandwidth of 16.6 The 750–840 nm output from the are usually accumulated together as a cm-1 is calculated. regenerative amplifier can be manipulat- negative time delay between the pump

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FIG.6. Schematic representation of an oscillator/amplifier based picosecond time-resolved resonance Raman setup. and the probe pulse, i.e., the probe multi-kilohertz 10 W lasers outputs. rescence stemming from the use of laser arrives at the sample first. At this point it Briefly the laser system is based upon wavelengths that match an electronic is appropriate to mention that in the field a 10 kHz synchronized dual-arm femto- excitation is considered to be a major of time-resolved spectroscopy, each second and picosecond laser system. obstacle in obtaining a background-free time-resolved spectroscopic technique Ultrafast high-average-power titanium neat resonance Raman spectra. The gives unique information, each set of sapphire lasers and optical parametric highly intense inherent fluorescence equipment is optimized to one tech- amplifiers provide wavelength tuning masks the weaker Raman signatures. A nique, and each fulfills the shortcomings across the ultraviolet (UV) to the mid- two-way approach has been developed of another; hence, all techniques are infrared. Of course, such a powerful and over the decades. In the first place the complementary to one another. Recently versatile laser system requires a wide fluorescence can be avoided by using an a new generation of time-resolved laser range of detectors and for this, custom- excitation line in the fluorescent free spectrometer systems has appeared with ized silicon, indium gallium arsenide, DUV, UV-C, and NIR regions of the versatility in mind capable of perform- and mercury cadmium telluride linear electromagnetic spectrum. However, ing time-resolved absorption, fluores- array detectors are essential to monitor this approach limits access to the cence, IR, and resonance Raman the probe laser intensity from the UV to resonance condition, as a maximum experiments, fsSRS, 2DIR, and TR- mid-infrared, monitoring direct scatter- number of probes absorb in the UV 2DIR across the microsecond to femto- ing or relative changes in sample and visible region. A better approach is second time regime.64 absorbance (DOD) of 10-5 in 1 sec- to deal with fluorescence by developing The ULTRA laser system plays on the ond.65 experimental means so that any fluores- strength of recent advances in cryogenic The Experimental Solutions for cent sample can also be probed by technology to control and stabilize Fluorescence Rejection. Sample fluo- resonance Raman spectroscopy.

1096 Volume 65, Number 10, 2011 Whilst surface-enhanced Raman spec- when closed, the capability of transmit- sion process) is blocked. Figure 7B troscopy66 is one of the better known ting the Raman spectral range, not shows the experimental setup for per- techniques to quench fluorescence (by adding additional noise to the measure- forming Kerr gated TR3 spectroscopy the effect of surface plasmons), it has ments, and not perturbing the positions and its application of fluorescence limited application to TR3 spectroscopy and widths of Raman bands. Deffon- rejection from a highly fluorescent given the metal surface present. Sample taine et al. demonstrated this for ground- sample, gasoline. The Kerr gate has fluorescence can be reduced (quenched) state Raman measurements using a 25 ps successfully been combined with by adding a ‘‘quencher’’ but this may run gating pulse and a carbon disulfide Kerr SERDS for effective fluorescence back- into problems if the Raman signature gate, although no Raman spectra were ground rejection.78–82 The application of from the ‘‘quencher’’ interferes with the reported.77 Using a 1 ps gate pulse it has Kerr gated Raman has generated several sample’s Raman spectrum. been possible to achieve a 3 ps time milestones over the last decade and a Important techniques used to date for response capable of efficient fluores- particular highlight has stemmed from fluorescence rejection include shifted- cence rejection. its application to investigations of solid excitation Raman difference spectrosco- Experimentally the optical Kerr gate materials. py67,68 (SERDS), subtracted shifted consists of two crossed polarizers and a The Kerr gate was able to resolve the Raman spectroscopy (SSRS), polariza- Kerr medium that is activated to prop- timescales for Raman photons to emerge tion modulation,69 shifted spectra,70 agate the light of interest by an intense from deep within diffusely scattering Fourier transform filtering,71 and tem- laser pulse polarized at 458 with respect materials. In essence, longer time delay poral gating.72 to the Raman and fluorescence light. A between the ps Raman pulse and In the case of SERDS, two individual schematic diagram is shown in Fig. 7. opening of the Kerr gate means that Raman spectra used to be recorded with As the gating pulse interacts with the one can collect Raman photons generat- two close-lying excitation lines (wave- Kerr medium, it induces a transient ed at greater depths from the sample. length difference will be in the range of anisotropy through a third-order nonlin- The technique demonstrates the ability 0.001 nm). The subtraction of one ear effect. When the gate is active the of time-resolved Raman spectroscopy to spectrum from the other can provide a two components of the electric field of assess the quality of bone transcutane- fluorescence-free Raman spectrum. In the passing signal are retarded with ously, measuring both the inorganic and this case, the fixed pattern noise will be respect to each other, transforming the organic content of bone.83 The applica- in the same position and is also removed light polarization from linear to ellipti- tion of Kerr gated Raman not only by subtraction. For SERDS a tunable cal. The propagation length through the provided a means to temporally resolve laser (dye laser or OPO) is the prime Kerr medium or the strength of anisot- collagen signals in the skin from colla- requirement. ropy can be chosen so that the polariza- gen signals in the bone beneath the skin The temporal gating technique is one tion of the Raman light is transformed but also showed how the relative of the rapid methods that employs back to being linearly polarized but spectral band intensities for the mineral simultaneous recording of signal over a rotated by 908 with respect to its original and organic spectra measured through long range of wavelengths to reduce or polarization direction. The dependence the skin in two mice genotypes were reject fluorescence from Raman scatter- of the induced refractive index change, different. In a study of oim (osteogenesis ing. In this technique the Raman spec- Dn, on the intensity of the gating pulse, imperfecta model) mice, the band of the trum is acquired by a short pulse laser I, is given by Eq. 3, where n2 is the mineral phase (phosphate m1, observed (~1 ps) and the long-lived (several ns) nonlinear refractive index of the Kerr at 958 cm-1) was smaller in oim/oim fluorescence background is removed in medium. (osteogenesis imperfecta model) mice the time domain. Gated ICCDs73 and compared with the CH wag of collagen Dn ¼ n I ð3Þ 2 streak cameras74 are the detectors pop- 2 of the organic phase (1451 cm-1). The ularly used in the nanosecond as well as In effect the medium acts as a transient oim mouse fails to synthesize functional ultrafast time domain for temporal k/2 waveplate. For this the gating pulse pro alpha 2(I) collagen chains, synthe- fluorescence rejection. An alternative energy, E (assuming flat-top temporal sizing only homotrimers of pro alpha uses the availability of high-power lasers and spatial profile), is dictated by Eq. 4: 1(I) collagen chains. This leads to poor to produce a third synchronized laser mineralization in bone tissues and the 2 pulse in addition to the pump and probe E ¼ pd kt=8Ln2 ð4Þ observed lesser phosphate band intensity TR3 beams, which can be used to drive The work spawned lateral thinking and a Kerr gate.75,76 where d is the gating pulse diameter in the team went on to invent spatially Ultrafast TR3 Spectroscopy Using the Kerr medium, k is the wavelength of offset Raman spectroscopy81 and exploit Ultrafast Kerr Gating. The optically the passing Raman light, t is the gating the technique commercially.84 The driven Kerr gate fulfills the essential pulse duration, and L is the length of the above ideas are now successfully used criteria for rejecting fluorescence from Kerr medium. The rotated light is then by other research groups as well.85 resonance Raman spectra. These criteria transmitted through the cross-polarizer Temperature (T)-Jump Raman Ex- include the need for a fast gating time, onto the spectrometer slit where the periment. In the laser T-jump Raman the efficient transmission of Raman unrotated component, in this case the experiment, the temperature jump within signals, blocking fluorescence efficiently fluorescence (which is a delayed emis- a defined time period is generally

APPLIED SPECTROSCOPY 1097 focal point review

FIG.7. (A) Detailed beam paths for sample collection and steering optics used for Kerr-gated TR3 and picture of lab setup. (B) Schematic presentation of setup for ps Kerr-gated TR3 spectroscopy. (C) Demonstration of Kerr gate technology using a gasoline sample (lRaman = 400 nm (1 ps, 4 mJ, 1 kHz)/400 s). (a) Attempt without Kerr gate activated, seeing only fluorescence, (b) fluorescence curve in (a) fitted to polynomial baseline and subtracted to show Raman spectrum is lost in the noise, (c) Kerr gate activated and a clean resonance Raman spectrum observed. NOTE: different intensity scales for (a, b and c).76 [Reprinted from P. Matousek, M. Towrie, C. Ma, W. M. Kwok, D. Phillips, W. T. Toner and A.W. Parker, J. Raman Spectrosc. 32, 983 (2001). Copyright 2011 John Wiley & Sons.] created by heating the aqueous solvent protein folding is generally sensitive to designed for study of protein folding, medium using a NIR laser source. The temperature and also occurs on the is shown in Fig. 8. NIR sources in the wavelength range of microsecond time scale. So by indirectly 1500–2200 nm that are typically em- heating water, a condition of T-jump of PROCEDURE FOR ployed are produced by pumping gas certain uniform size and duration is SYSTEMATIC Raman shifters with the fundamental created that satisfies the requirement to INTERPRETATION OF TR3 frequency (1064 nm) of Nd:YAG la- probe the intermediate states during SPECTRA sers.86,87 This range of NIR wavelengths folding, unfolding, or misfolding. A is important as water absorbs strongly in typical T-jump profile produced from The combined experimental and the- this region, providing a means to rapidly the NIH setup is shown in Fig. 8. With oretical approach is necessary for com- transfer energy into the medium and so the initial trigger using the T-jump plete spectral and structural analysis of produce the required temperature in- mechanism the RR spectrum can be short-lived intermediates observed in the crease. recorded in the UV and visible regions. ps–ls domain. The approaches include The T-jump experiments are interest- To date the time resolution of T-Jump ing from the perspective of biochemical achieved is reported in the picosecond (1) Correlation of experimentally ob- processes that occur in the microsecond range.88 A typical ns T-jump-UVRR served Raman bands to a known to nanosecond time frame. For example, experimental setup at Asher’s lab,89 group of functional groups compar-

1098 Volume 65, Number 10, 2011 vibrational modes can be divided into IR active and Raman active vibrations. This approach usually helps in eliminating many calculated Raman inactive modes. (4) Measurement of depolarization ratio and Raman excitation proﬁle (REP) of corresponding Raman bands. In the computational process the vibration of a molecule is described by the quantum harmonic oscillator and the calculated harmonic frequencies are typically larger than the experimentally observed frequency. This is mainly due to the fact that the anharmonic effects are not included in the theoretical method. The overestimated calculated theoretical frequencies are always mul- tiplied by a correction factor (scaling factor) for better agreement with the experimental frequencies. If the scaling factor does not give uniform agreeable frequency then a common scaling factor must be calculated.92

APPLICATIONS OF TR3 SPECTROSCOPY Time-resolved resonance Raman spectroscopy in the fast and ultrafast time domains has successfully been applied to various systems of chemical and biological importance for studying structure, reactivity, and dynamics of excited states and reactive intermediates. In this section we briefly describe a few FIG.8. (A) A typical T-jump profile for T-jump Raman experiment.87 [Reproduced from J. of the photo-processes involving model Kubelka, Photochem. Photobiol. Sci. 8, 499 (2009) by permission of The Royal Society of organic, inorganic, and biological sys- Chemistry (RSC) for the European Society for Photobiology, the European Photochemistry tems, explored by TR3 spectroscopy. Association, and the RSC.] (B) The T-jump spectrometer consists of a Nd:YAG laser (YAG), Organic Model Systems. Aromatic two H2 Raman shifters (R1 and R2), a thermostated flow cell sample circulator (FC), and a Spex Triplemate triple monochromator with a blue-enhanced ICCD detector (SP). The Quinones. Quinones hold a special heating pump beam is obtained by Raman shifting the YAG fundamental in H2 to 1.9 lm place in the development of TR3 (first Stokes). The probe beam is obtained by Raman shifting the YAG third harmonic to 204 spectroscopy. Academically this class nm (fifth anti-Stokes). The time delays between the pump and probe pulses are provided by the variable delay line (DL).89 [Reprinted with permission from I. K. Lednev, A. S. Karnoup, M. of compounds is an integral component C. Sparrow, and S. A. Asher, J. Am. Chem. Soc. 121, 8074 (1999). Copyright 1999 American of the photosynthetic reaction centers Chemical Society.] and plays a major role in electron transport in light-induced photosynthesis and aerobic respiration. The mechanistic ing similar known systems and Moller Plesset (MP), and density 90,91 pathways for photosynthesis involve a isotopically substituted systems. functional theory (DFT) with a large number of intermediate steps, such as (2) Generating TR3 data that is capable number of basis sets provide sup- photoinduced electron transfer, proton of providing qualitative data about porting quantitative structural data transfer, and hydrogen atom transfer structure (increase/decrease in bond as well as vibrational frequencies. reactions that exploit the reversible length, bond angle, or dihedral The band assignment used to be redox properties of quinones. Therefore, angle). The measurement is not made on the basis of comparison of it is important to study the excited-state quantitative in nature for defining calculated and experimentally ob- RR spectroscopy of model quinone structural parameters. served frequency. For nonlinear systems, mimicking a few reaction steps (3) Combining Hartree–Fock (HF), molecular systems, the 3N - 6 occurring in photosynthetic reaction

APPLIED SPECTROSCOPY 1099 focal point review

FIG.9. Chemical structures of model quinones studied by TR3 spectroscopy.

centers. Figure 9 gives molecular sche- of pump pulse on the sample, the S0 substituted BQs is different from BQ matics of a few of the model quinones state is excited to higher Sn states that and the reactivity is highly dependent on studied to date by TR3 spectroscopy. relax through different pathways de- functional group substitution and the Tripathi and co-workers have studied pending on the nature of the medium. nature of the solvent medium. The ns anion radicals of several quinones by A ps TR3 study of BQ in H2O:alcohol TR3 spectroscopic study of ubiquinone pulse radiolysis based TR3 spectrosco- mixed solvents shows the formation of (UQ) using a 248 nm pump source in 2,3 py. Parent benzoquinone (BQ) ab- the semiquinone radical (BQH )asa H2O, ethanol, and cyclohexane suggests sorbs at around 250 nm in the ground result of fast hydrogen atom transfer hydrogen transfer reaction from solvent state. Therefore, 248 nm or 266 nm from the solvent to the triplet state of to the lowest triplet state and shows radiation from any commercial laser BQ, which is formed via vibrational different reactivity pathways towards source is the best choice for use as the relaxation of the Sn state in 20 ps after various reactive intermediates. Using pump laser source to create the excited- the photolysis.93 the probe source of 440 nm from a dye state intermediates. With the incidence The excited-state photochemistry of laser, the triplet state formed in an

1100 Volume 65, Number 10, 2011 aqueous medium was observed to oxi- dize water molecules and undergo a photoinduced electron transfer (PET) reaction to form a UQ radical anion (UQ-). In ethanol medium, the simultaneous formation of radical anion and semiquinone radical (UQH) was observed as a result of probable PET and hydrogen atom transfer reactions.6 The identiﬁcation of Raman bands of the radical anion were further supported by probing the reaction of triplet UQ (3UQ) with sodium nitrite as electron donor that directly forms the radical anion. The rate of hydrogen transfer reaction depends primarily on the hydrogen donating ability of the solvent. In the less- reactive solvent cylohexane the triplet (np*) state is observed via intersystem crossing (ISC) from the singlet state. The TR3 bands and structural details for the triplet state, radical anion, and semiquinone radicals were assigned (notably for C=O and C=C) on the basis of comparison with the theoretical and the experimental data obtained for normal and isotopically substituted parent BQ. From the calculated94,95 and experimentally observed RR bands of UQ, unlike benzoquinone where the coupling of C=O and C=C modes were observed, the two stretching CO modes were found to be decoupled. Figure 10 presents the experimentally observed Raman signature of intermediates in different solvents. This particular experiment extracts structural information along with the kinetics of differing pathways of formation and the decay of each individual intermediate in various solvents with a high accuracy and demonstrates the advantage of TR3 spectroscopy over other time-resolved techniques such as TRA and time- resolved ﬂuorescence (TRF). Similarly duroquinone (DQ) is anoth- FIG. 10. The ns TR3 spectra of UQ (10-3 mol dm-3), (A) in ethanol, (a) no acid present, (b) er methyl-substituted model quinone small amount of acid added to (a), (c) NaNO3 added to (a), relative intensity divided by 3. Spectra (a–c) have the probe-only spectrum subtracted; time delay between pump and that is considered to be a powerful probe laser 50 ns. (d) probe-only spectrum with solvent bands removed, (B) in water (a) pH 6 one-electron oxidant. (The reduction and (b) pH 2, adjusted by addition of HCl. Time delay between pump and probe laser 80 ns, potential of (3DQ/DQ-)isþ2.17 V.) probe-only spectra subtracted from both spectra and (C) in cyclohexane, pump/probe time The ns TR3 experiment was performed delays (a) probe only, (b) 100, (c) 50, and (d) 25 ns. The beauty of TR3 techniques is the way they provide spectroscopic differentiation of the relatively similar chemical intermediates.6 by using 355 nm as pump wavelength [Reproduced from A. W. Parker, R. E. Hester, D. Phillips, and S. Umapathy, J. Chem. Soc. and 425 nm and 500–520 nm as the Faraday Trans. 88, 2649 (1992) by permission of The Royal Society of Chemistry.] probe source for investigating the semiquinone radical and triplet state, respectively.96 The above probe wavelengths are selectively in resonance with the proposed intermediates as derived from

APPLIED SPECTROSCOPY 1101 focal point review

formation of semiquinone radical FA in isopropanol. Time-resolved resonance Raman spectroscopy of bromanil in noninter- acting CCl4 solvent at different time delays using 355 nm and 512 nm as pump and probe source, respectively, yields the lowest excited triplet state of pp* character. Along with the triplet state, the semiquinone radical and radical ion were also formed after photolysis at different environments.102–105 RR bands were assigned by comparing the vibrational modes of the ground state and intermediate states of related species. As observed in the case of SCHEME 2. Probable mechanism for bimolecular hydrogen abstraction reaction of FA.100 fluoranil, the effect of substitution of [Reprinted from G. Balakrishnan and S. Umapathy, Chem. Phys. Lett. 270, 557 (1997). the bromine atom is more pronounced in Copyright 1997 Elsevier]. the excited state than in the ground state, as the symmetry of the molecule in its excited state is distorted compared to the 96 the laser flash photolytic study. In the 1,4-naphthoquinone) probed in the ground state. The structure of the radical 97 non-interacting solvent acetonitrile, the nanosecond domain using 266 nm anion is also altered in a similar fashion triplet-state formation is favored, having and 416 nm as pump and probe show and the largest structural reorganization a lifetime in the range of a few considerable coupling between C=O was observed for the semiquinone microseconds. In the presence of a and C=C stretching frequencies. The radical. hydrogen donating solvent the Raman importance of asymmetric substitution Vitamin E: A Tale About the Real- signature for durosemiquinone radical in the case of MQ is highlighted by Time Functionality of Antioxidants. becomes prominent, indicating that the analyzing the important Raman signa- An antioxidant is a molecular system triplet state is the precursor for forming ture. The TR3 study on the intermediate that inhibits oxidation of other mole- the semiquinone radical. Triplet forma- radical ion of methyl-1,4-benzoquinone cules. For example a-tocopherol (Fig. tion was further confirmed by monitor- and 2,6-dimethyl-1,4-benzoquinone pre- 11A), the main component of vitamin E, ing the kinetics in the presence of sents an example of structural distortion is a biological lipophilic membrane oxygen, which quenches the triplet state, with the change in substitution.98 component and a chain-breaking antiox- forming singlet oxygen via an energy Perhalo (chloro, fluoro, and bromo) idant. Its role is to protect cellular transfer mechanism. The strongly oxi- derivatives of BQ are important model membranes against lipid peroxidation, dizing nature of quinones can also be systems to study several photochemical a process similar to that of linseed oil studiedbyaddingareductant.The and photophysical processes. The effect drying. The a-tocopheroxyl radical re- quinone radical anion may be studied of halogen substitution on reactivity of sulting from the breaking of this radical by exciting the quinone in the presence perhalo BQs was exploited using TR3 chain reaction is extremely stable, and of the reducing agent sodium nitrite. The spectroscopy. Fluoranil (FA) in the under certain conditions, for example in triplet DQ reacts with suitable antioxi- lipid micelles, it can have a lifetime of presence of hydrogen-donating solvents dants (a-tocopherol and 6-palmitoyl-L- up to a few minutes. Under biological such as isopropanol effectively under- ascorbate) at a diffusion-controlled rate. conditions vitamin E is believed to be Again the same reaction in a micellar goes a bimolecular hydrogen abstraction 99–101 rejuvenated by the much larger pool of medium was proven to be initialized by reaction in a radical fashion in its ascorbate acid (vitamin C), as shown hydrogen atom transfer. The motivation lowest triplet state to form a ketyl Fig. 11B, although this is controversial for studying such fundamental processes radical. The assignments of Raman for reasons we now discuss. in different environments (nature of bands were performed by taking theo- The interactions between vitamin E solvent, antioxidants, surfactants, mi- retically obtained frequencies into ac- (main component, a-tocopherol, Fig. celles, etc.) is to mimic the naturally count and comparing other perhalo BQs 11A) and vitamin C (ascorbate) have occurring photoprocesses and identify (chloranil) and BQ. It was observed that been of considerable interest and spec- the best set of chemically engineered the perfluoro effect is more pronounced ulation106 since the first direct observa- model systems having photochemical in the excited state and as a result the tion using fast reaction methods of the behavior and reaction parameters that extent of delocalization in the anti- repair of the vitamin E radical by best approximate those found in nature. bonding p* orbital is reduced in the vitamin C in solution by Packer et The radical ions of naphthoquinone case of fluoranil over BQ. Scheme 2 al.107 in 1979 and by Bisby and (NQ) and menaquinone (MQ, 2-methyl- represents the possible mechanism for Parker108 in membrane systems in

1102 Volume 65, Number 10, 2011 FIG. 11. (A) The structure of a-tocopherol (vitamin E). (B) (a) The photoionization process to generate the a-tocopheroxyl radical. (b and c) The equation for the antioxidant quenching of lipid free radicals and proposed reformation of route of a-tocopherol by vitamin C ascorbate (ASCH). (C) Resonance Raman spectra of (a) a-tocopheroxyl, (b) b-tocopheroxyl, and (c) d-tocopheroxyl radicals obtained by photolysis (308 nm) of the respective tocopherols (5 mmol dm-3) in methanol. The probe pulse was 425 nm, delayed 2 ls after the pump pulse.109 [Reproduced from A. W. Parker and R. H. Bisby, J. Chem. Soc. Faraday Trans. 89, 2873 (1993) by permission of The Royal Society of Chemistry.] (D) Proposed structure of tocopheroxyl radical. (E) Vitamin E–Vitamin C damage and repair. (F) Proposed reaction mechanism of antioxidant activity.96 [Reprinted with permission from R. H. Bisby and A. W. Parker, J. Am. Chem. Soc. 117, 5664 (1995). Copyright 1995 American Chemical Society.]

1995, including time-resolved Raman form to be relatively long-lived in vivo. increase (through incubation with dehy- studies.109 Despite this, there is less As a consequence, other bioreductants droascorbate) intracellular vitamin C reliable evidence of this interaction in of the vitamin E radical such as levels.111 The transient Raman signa- vivo. As the principal antioxidant in ubiquinol110 have also been proposed. tures of a-tocopheroxyl radical are given biomembranes and lipoproteins, a- The recycling of vitamin E by ascor- in Fig. 11C. These experiments utilized tocopherol is subject to constant oxida- bate has been demonstrated in erythro- resonance Raman and therefore required tive reactions, but it is known from the cytes through strategies that both two speciﬁc laser wavelengths, the ﬁrst studies of biokinetics of the deuterated decrease (by reaction with tempol) and to photoionize the vitamin E (308 nm)

APPLIED SPECTROSCOPY 1103 focal point review and the second tuned to resonance with triplet energy can’t be used effectively to and is lower than the ground-state the absorption of the radical species form singlet oxygen. In nonaqueous Raman band position at 1639 cm-1, (425 nm). Figure 11C illustrates the solvents 3DQ was observed to be indicating a weaker C=C bond in the resonance Raman spectra of a range of reduced by both antioxidants in a excited state.118 tocopheroxyl radicals. diffusion controlled rate. This rate is Figure 12B presents the ps-TR3 The two intense Raman features at comparable and in accordance to the spectra of olefinic C=C (left) high 1504–1490 cm-1 and 1595 cm-1 are indirectly obtained singlet oxygen lumi- wavenumber region and (right) low assigned to the CO stretch (Wilson m7a) nescence yield. The rate of the same wavenumber anti-Stokes and Stokes andringC–Cstretch(Wilsonm8A), reaction in the presence of SDS micelle region of S1 trans-stilbene in chloroform respectively. The high band intensities is governed by the rapid exit of 3DQ solution obtained at selected time delays are believed to be due to the structural from the micellar environment. The up to 100 ps.119 Close inspection of the properties of the vitamin E radical that scheme is depicted in Fig. 11F. Similar- olefinic C=C str. Raman signatures at attains a quinone-type structure as ly 3DQ reacts with antioxidant dihydro- different delay times shows the narrow- shown in Fig. 11D. Further studies of lipoate and lipoamide112 in electron ing of the 1570 cm-1 band and up-shift a-tocopheroxyl in different solvents transfer channels yielding durosemiqui- of band position at around 20–30 ps. with a wide range of polarity and none radical anion. It may be useful to Simultaneously changes were observed hydrogen bonding have found the 1595 mention here that reaction of different for the intensities in the anti-Stokes and cm-1 band to show more of a solvato- kinds of antioxidants can be tried with Stokes Raman bands in the low-fre- chromic effect than the 1504 cm-1 band. variable quinones (having a wide range quency Raman region. The relative The ratio of the intensities of the C–O of reduction potential) to understand the intensity of the Stokes to anti-Stokes stretch (m7a) and the m8A ring stretch generalized potential of antioxidants. band gives an idea about the tempera- intensity alters with solvent polarity. Mechanistic Insights for Excited- ture related to the vibrational mode as When a-tocopheroxyl is embedded in State Dynamics of trans-andcis- followed by the relation with the micelles the frequency of the m8A band Stilbene. Photoisomerization is a well- Boltzmann factor exp(-E/kT)whereE shows a downward shift (1593 cm-1 in studied unimolecular reaction, both trans signifies the vibrational energy level SDS and 1590 cm-1 in HTAC) that (E)tocis(Z)conversionandthe spacing under off-resonance condition. indicates a polar micellar medium, such converse. Both trans- and cis-stilbene Figure 12C shows the time-dependent as the polar solvents ethanol or metha- are archetypical examples for character- changes in the peak position for the nol. The corresponding values in the izing the dynamics of isomerization and olefinic band at 1570 cm-1,whichfitted presence of Triton-X (1586 cm-1) and in this section we briefly describe some to a single exponential decay with time DMPC (dimyristoylphosphatidylcho- facts and figures regarding the mecha- constant of 12 ps and thus can be used line) bilayers (1587 cm-1) signify a nistic investigations of this molecule as a spectroscopic thermometer for non-polar environment more akin to provided by TR3. following the energy relaxation of the hexane. Interestingly, performing the With UV photoexcitation to the first initial singlet excited state. The anti- experiment in DMPC micelles better singlet excited state (S1), the structural Stokes resonance Raman spectra of S1 mimics the mammalian membrane and rearrangement of twisting around the trans-stilbene in n-hexane were ob- gives an intensity ratio commensurate central olefinic C=C bond has been tained using 290–310 nm pump and with a semi-polar environment, suggest- observed.113 At room temperature the 580–620 nm probe with 8 ps temporal ing that once the a-tocopheroxyl radical theoretically estimated barrier for pho- resolution.120 All the dominant band is formed it moves to the edge of the toisomerization and experimentally ob- positions observed in the Stokes spec- aqueous/micelle interface, a place where served lifetime for S1 trans-stilbene in a trum are clearly visible as well in the it can readily interact with vitamin C, low viscosity solvent was found to be anti-Stokes spectrum. The intensity of indicating that in our cells the precious around 1000 cm-1 and 70 ps, respec- the anti-Stokes signatures was observed vitamin E would be rejuvenated and tively.114,115 The first TR3 details of to rapidly decrease on a 10 ps time empowered to continue its vital role as a trans-stilbene were reported indepen- scale. The dynamics being visualized in chain-breaking antioxidant as shown in dently by the Gustafson116 and Hama- both the anti-Stokes and Stokes win- Fig. 11E. guchi groups.117 The experimentally dows were similar. The study of the reaction between observed TR3 spectra in n-hexane using In contrast to trans-stilbene, the triplet duroquinone (3DQ) with antioxi- 266 nm pump and 585 nm probe,117 photoisomerization of cis-stilbene oc- dant a-tocopherol and ascorbate was power dependence behavior, and Raman curs from the first excited electronic studied using laser flash photolysis and excitation profiles (REP) clearly suggest state as a barrierless transition and 96 ns TR3 spectroscopy. The idea behind the formation of S1 trans-stilbene within completes on a 1 ps time scale and thus this study was to evaluate quinone the time resolution of the experiment. is able to effectively compete with systems (duroquinone and ubiquinone) Experimental spectra from both groups vibrational relaxation. The position and as singlet oxygen sensitizers in the constitute several strong Raman bands at the width of the Raman bands provide presence of the antioxidants. In other 1144, 1177, 1238, and 1570 cm-1 as the frequencies and dephasing time of words if the triplet energy of 3DQ can be shown in Fig. 12A. The 1570 cm-1 vibrational motions in the excited state. quenched by the antioxidants, then the band was attributed to olefinic C=C str. There have been few studies of cis-

1104 Volume 65, Number 10, 2011 stilbene using TR3 spectroscopy due to short lifetime and inaccessible pump (260–290 nm) and probe (630–670 nm) wavelengths. The ﬁrst ps TR3 spectra of cis-stilbene in n-hexane using 267 nm pump and 630 nm probe was reported by the Rutherford Appleton Group (RAL group).121 Intense Raman bands at 243, 475, and 742 cm-1 were observed for the TR3 spectrum as depicted in Fig. 12D. The spectra obtained in different solvents, at longer pump and probe wavelengths, all gave a similar result except that the measured lifetimes differ slightly. As the laser pulse duration was ~ 1 ps, the lifetime of excited S1 cis-stilbene was estimated to be around 1.5 ps. Further, unlike trans-stilbene, cis-stilbene does not show any pump and probe laser power dependence, implying the spectra are not due to a nonlinear process. The observed 243 cm-1 band was assigned to a mixture of CC torsion, in-plane bending, and ethylenic torsion. Further, the ps TR3 study on deuterated derivatives of cis-stilbene in n-hexane showed the existence of resonantly enhanced dominant second-order progression originat- ing from the 229 cm-1 fundamental band.122 Charge Transfer in 4-Dimethylami- nobenzonitrile (DMABN) and other Substituted Benzonitriles. Over four decades since its discovery, DMABN, an intramolecular donor–acceptor molecular entity, has been the subject of

FIG. 12. (A) Transient resonance Raman spectra of trans-stilbene in n-hexane (2 3 10-3 M). (a) Difference spectrum (b) - (c); (b) spectrum obtained with both the pump and probe lasers incident on the sample; (c) spectrum obtained with the probe laser only.117 [Reprinted from H. Hamaguchi, C. Kato, and M. Tasumi, Chem. Phys. Lett. 100, 3 (1983). Copyright 1983 with permission from Elsevier.] (B) High and low wavenumber Stokes spectra of trans-stilbene. (C) Time dependence of 1570 cm-1 Raman bands; the Raman thermometer119 [Reprinted with permission from K. Iwata and H. Hamaguchi, J. Phys. Chem. A 101, 632 (1997). Copyright 1997 American Chemical Society.] (D) ps TR3 spectra of (a) cis- and (b) trans-stilbene at 0 ps and 20 ps delay time, respectively.121 [Reprinted from P. Matousek, A. W. Parker, D. Phillips, G. D. Scholes, W. T. Toner, and M. Towrie, Chem. Phys. Lett. 278, 56 (1997). Copyright 1997 with permission from Elsevier.]

APPLIED SPECTROSCOPY 1105 focal point review

parameters involved. These results support the TICT model. In non-polar CCl4 solvent, fluorescence quenching of DMABN was observed. The results from fs fluorescence and ps and ns time-resolved Raman techniques suggests an intermolecular electron transfer between the LE state and the solvent that occurs on a time scale of 500 fs and leads to formation of the DMABN cation radical.134 The DMABN–Cl adduct is formed at a time constant of 13 ps and has a lifetime longer than 50 ns. The structural analysis of the adduct predicts the preferable position of the Cl atom at the amino para ring position of the DMABN radical cation. The adduct possesses a relative planar conformation for the dimethylamino group with respect to the phenyl ring. Figure 13 shows the ps Kerr-gated SCHEME 3. Chemical structure of DMABN and the schematic chemical representation of TR3 spectra obtained for the ICT state in different types of ICT. methanol solvent of DMABN and its isotopic analogues, which play an es- intense investigations driven by the need sential role in explicitly analyzing and to understand intermolecular electron transfer and the role the solvent plays in mitigating the process.123 DMABN became a highlight when it was noted to have a solvent-dependent fluorescence spectrum that was dependent on whether it was present in a non-polar or polar solvent. It has been hypothesized and proven that in non-polar solvents the observed fluorescence is from a locally excited state (LE). In polar solvent the LE state forms a stable intramolecular charge transfer (ICT) state by nonradiative interconversion that contributes toward a second fluorescence band. The LE to ICT conversion is favored in polar solvents. TR3 has been shown effective in interrogating the structural details of these intermediate states in solution phase and the mechanism of charge transfer reactions. The structure of the ICT state has been fiercely debated and falls broadly into four categories as described in Scheme 3. Twisted ICT (TICT),124 wagged ICT (WICT),125 planar ICT

126 15 (PICT), and rehybridization ICT FIG. 13. (A) TR3 spectra of ICT state of DMABN (ground state S0) and (ICT), DMABN-N , 127 (RICT). Time-resolved vibrational and DMABN-d6 in methanol (267/330 nm pump/probe, 50 ps delay time, methanol solvent, 133 (IR128–130 and RR131–133) spectroscopy 100 min acquisition time). [Reprinted with permission from W. M. Kwok, C. Ma, P. Matousek, A. W. Parker, D. Phillips, W. T. Toner, M. Towrie, and S. Umapathy, J. Phys. Chem. has been used to derive the character of A 105, 984 (2001). Copyright 2001 American Chemical Society.] (B) The table gives the ICT state along with the structural calculated shifts and TR3 observed shifts of the Raman bands for the various structures.

1106 Volume 65, Number 10, 2011 tence of two charge transfer states, namely ICT and hydrogen bonded ICT (HICT), in equilibrium.136 The equilibrium between the populations of both states was observed at a time constant of 13 ps. The nonradiative internal conversion, the de-excitation rate, of the HICT state was found to be much larger, which results in reduction of the fluorescence quantum yield. It is interesting and important to observe such variation in excited-state structures with minimal change in solvent polarity, which greatly impacts reactivity and thus quantum yield of fluorescence. The development of the ps Kerr-gated TR3 technique enabled better understanding of the dynamics of highly fluorescent molecule such as DMABN. It is repetitive but nonetheless necessary to state that the TRA, TRIR, TRF, and TR3 data obtained from DMABN in different solvents are purely complementary in nature and in combination bolsters the already known and newly found mechanisms for excited-state dynamics. The combined TR techniques of ps Kerr-gated TR3, TRIR, and TRA can be applied to other benzonitrile systems such as 4-diethylaminobenzonitrile (DEABN) and 4-dimethylamino-3,5-di- methylbenzonitrile (TMABN) as well as DMABN to predict the variation in dynamics with functional group substitution.137 Figure 14 shows the ps TR3 spectra of all the mentioned systems in acetonitrile solvent, each possessing distinct features for the ICT of each molecule. The formation of the ICT state of FIG. 14. Picosecond Kerr-gated time-resolved resonance Raman spectra of (a) DMABN, TMABN was observed to be faster than (b) DEABN, and (c) TMABN in acetonitrile obtained with 267 nm pump and 400 nm probe the others and forms immediately after wavelengths at 50 ps time delay. The intermolecular charge transfer (ICT) reaction is fastest for TMABN, thus supporting the concept that ps-K-TR3 spectra TICT states are all similar, the electronic excitation. The formation with frequencies resembling those of the benzonitrile radical anion.137 [Reproduced from W. of the ICT state for DEABN was M. Kwok, C. Ma, M. W. George, D. C. Grills, P. Matousek, A. W. Parker, D. Phillips, W. T. Toner observed to be faster than that for and M. Towrie, Phys. Chem. Chem. Phys. 5, 1043 (2003) by permission of the PCCP Owner DMABN. The spectral data obtained Societies.] for all the above benzonitrile systems favor the TICT model. assigning the vibrational bands. The at the lowest triplet state. The experi- Biological Systems. Understanding pump and probe wavelengths were 267 mental and theoretical structural analysis of the Photocycle of Bacteriorhodopsin nm and 330 nm, respectively. Unlike the of the lowest triplet state of DMABN and Resolving the Baby Steps Towards ultrafast domain, the triplet-state RR suggests a planar or near-planar struc- Biological Functionality. TR3 spectros- spectral study used ns TR3 spectroscopy ture with high localization of negative copy in the nanosecond and picosecond and this state shows no significant charge on the cyano group along with a domain has successfully been applied difference between polar and non-polar substantial change in the ring skeleton. for further understanding the photo- solvents.135 Thus it was possible to Ultrafast time-resolved IR study of chemical pathways of many complex conclude the existence of only one form DMABN in methanol predicts the exis- light-initiated biological phenomena.

APPLIED SPECTROSCOPY 1107 focal point review

photobiological-active sites can be visualized. We turn first to the structure and reactivity of the various intermediates that appear and disappear during the photocycle of bacteriorhodopsin (bR). Bacteriorhodopsin (bR) is a notable retinal protein complex found in cell membranes of Halobacterium halobium, which basically acts as a proton translo- cating system, moving protons across the membrane and out of the cell by creating a proton gradient. The all-trans retinal is a light-sensitive chromophore attached to a lysine group through a protonated Schiff’s base, lying in the heart of bR, responsible for its photoac- tivity through a photocycle. Figure 15A depicts the structure of all trans-retinal along with various short-lived reactive intermediates formed during the photocycle.138–144 Along with RR spectroscopy, other spectroscopic and biochemical techniques have also played their parts and all together are responsible for elucidating the complex intermediates that require the resolution of femtosecond to millisecond response. Light absorption by the all-trans retinal chromophore isomerizes it at the C13=C14 double bond to produce a cis form that triggers a series of reactions. Using ps TR3 spectroscopy with 3 ps time resolution (550 nm pump and 589 nm probe) Mathies’ group144 identified the J intermediate, which is thermally excited and has a twisted structure with intense hydrogen-out-of-plane (HOOP) modes at 956 cm-1 and 1000 cm-1 and broad fingerprint signatures in the region of 1100–1200 cm-1. The instrument was specifically developed to record both Stokes and anti-Stokes data on ultrafast time scales. The HOOP vibrational mode is a signature of twisted conformers and its absence indicates that the structural change leads to a planar FIG. 15. (A) The bacteriorhodopsin photocycle. (B) The summary of the structural changes conformer. In 3 ps, the intensity of the for bacteriorhodopsin intermediates. (C) Comparison of the J, K, and KL spectra found at ambient temperature with those of the photoproduct trapped at 77 K (left trace) and anti- HOOP modes decreases and the broad Stokes resonance Raman spectra of bacteriorhodopsin and its photoproduct for time delays fingerprint region collapses into a single from 0 to 10 p (right trace).143 [Reprinted with permission from S. J. Doig, P. J. Reid, and R. A. mode of vibration. Mathies, J. Phys. Chem. 95, 6372 (1991). Copyright 1991 American Chemical Society.] This phenomenon is indicative of the fact that the J to K isomerization is completed within 3 ps. The non-exis- Detection and vibrational analysis of photo- or air-sensitive and in addition tence of HOOP bands for the K stage macromolecular biological reactive in- difficult to obtain. The selectivity and suggests that the chromophore has termediates are more complex than the specificity of RR spectroscopy has relaxed about the single or double bond. chemistry of small organic entities and provided exactitude in the face of these Again, by 100 ps the HOOP band often biological materials are very difficulties, simplifying how complex reappears, suggesting a twisted structure

1108 Volume 65, Number 10, 2011 for the newly formed KL intermediate. Further spectra were obtained at various delay times between 0 ps and 13 ns in order to study the structural aspects of the intermediate. The spectral transition at various delay times that are directly related to the structure are shown in Fig. 15C. The anti-Stokes spectra obtained at various delay time are also depicted in Fig. 15C. The spectrum at 0 ps indicates a vibrationally hot species produced mostly due to the J intermediate state. The 1521 cm-1 band is assigned to ethylenic str. and decays by 2.5 ps. From the lifetime of the anti-Stokes bands (2.5 ps) it is clear that the transition between the vibrationally hot J stage and the relaxed, more planar K stage occurs preferably via vibrational cooling. In summary, the transition between J ! K ! KL stages occurs via a hot vibrational state ! relaxation via vibrational cooling ! reappearance of HOOP mode. A recent femtosecond stimulated Raman spectroscopic study by the same group138 in the sub-picosecond time domain suggests new findings but also confirms the already established experimental evidence. The new findings show that the appearance of the J stage is delayed by 150 fs with respect to zero time and has a rise time constant of 450 fs. Figure 15B summarizes the experimentally observed intermediates formed within the photocycle. Exploring the Ultrafast Dynamics of Inorganic Metal Complexes. Metal complexes are essentially inorganic

FIG. 16. (A) The typical relationships between the reactive excited states and the nature of the direct photoreactions in coordination compounds.145 [Reproduced from G. Stochel, M. Brindell, W. Macyk, Z. Stasicka, and K. Szacilowski, Bioinorganic Photochemistry (John Wiley & Sons, Chi- chester, UK, 2009, p. 48, with permission of John Wiley & Sons.] (B) TR3 of [Ru(b- py)3]Cl2 recorded in (left) acetonitrile, kpump 400 nm and kprobe 350 nm and (right) in H2O at kpump 400 nm and kprobe 475 nm. (C) Overview of processes following excitation 2þ 151 of [Ru(bpy)3] to FC State. [Reprinted with permission from W. Henry, C. G. Coates, C. Brady, K. L. Ronayne, P. Matou- sek, M. Towrie, S. W. Botchway, A. W. Parker, J. G. Vos, W. R. Browne, and J. J. McGarvey, J. Phys. Chem. A 112, 4537 (2008). Copyright 2008 American Chemical Society.]

APPLIED SPECTROSCOPY 1109 focal point review molecular systems consisting of a metal The singlet 1MLCT state is short lived time is found to be invariant irrespective center coordinated by a number of and vibrationally hot in nature and of isotopic substitution in ligands or ligands through elements such as N, O, structurally similar to its ground state. substitution of other ligands (heterolep- S, and P that have a ligand field around Picosecond TR3 shows that the excited tic complexes). Considering the range of the metal atom. Commonly used metals population of the 1MLCT state is polarity of the solvents used, it can be include the transition (Pt, Pd, Cu, Rh, depleted by intersystem crossing (ISC) suggested that the solvent independence Ru, Pt, etc.) and lanthanide series in the and vibrational relaxation to a less of the excited-state behavior is not periodic table.145 energetic thermally equilibrated excited governed by solvent reorganization or TR3 spectroscopy in the picosecond (THEXI) triplet 3MLCT state. The vibrational cooling towards formation of and nanosecond time domain along with character, formation, and decay dynam- the THEXI–1MLCT state. The lifetime density functional theory methodologies ics of the THEXI state and the related in all solvents using various heteroleptic have successfully been applied to study structure changes occurring in the ultra- complexes was estimated to be ~ 20 ps. the excited-state structure and dynamics fast time domain following activation This observation coincides with earlier dealing with the parallel complex deac- have been a matter of intense scientific results showing that vibrational relaxa- tivation pathways of inorganic metal debate. In general, upon photoexcitation tion of 3MLCT was completed within 6 complexes. The types of transitions for a the metal center and ligand groups ps and is devoid of any change in line typical octahedral complex can be undergo simultaneous oxidation and shape by varying from aqueous to classified as: reduction, the metal-to-ligand transfer alcoholic environment.153 By comparing of the electron. The metal complex with many other ultrafast studies the (1) LC: Ligand centered: observed for tris(2,20-bipyridyl)ruthenium(II) mechanistic pathways for photoinduced free ligand. 2þ ([Ru(bpy)3] ) has been an interesting processes occurring in the case of (2) LMCT: Ligand-to-metal charge 2þ system by virtue of its transition metal [Ru(bpy)3] is summarized in Fig. transfer: ligand is oxidized and photochemistry and photophysics. 16C. It is important to mention here metal is reduced. The collective femtosecond time-re- that in the case of bipyridyl (bpy) ligated (3) MC: Metal centered: transition from solved absorption and fluorescence up- metal complexes the excited-state struc- non-bonding t2g to the anti-bonding 2þ conversion study of ([Ru(bpy)3] )by ture and dynamics are governed by bpy eg orbitals. various research groups147–150 led to the units and can be followed by observing (4) MLCT: Metal-to-ligand charge proposal of a model where the growth of the vibrational changes of the bpy wing. transfer: metal center is oxidized 3MLCT from the initially populated As seen in the case of various cyclo- and the ligand is reduced. 1MLCT FC state is essentially complet- triphosphazene ligands substituted with With the incident laser pulse excita- ed within 1 ps. The solution to the pendant 2,20-bipyridyl moieties, irre- tion, immediate relaxation processes dilemma of time lapse for formation of spective of dissimilarity in crystal struc- accompanied by various reactions occur the THEXI–3MLCT state was further ture packing and space groups, the at different time scales and are summa- addressed by McGarvey and co-workers cyclotriphosphazene unit has little effect rized in Fig. 16A. The deactivation by using Kerr-gated ps TR3 spectrosco- on the excited-state RR properties of the processes are dependent on the type of py with 3 ps temporal resolution.151 This Ru complexes.154 system under study, i.e., structure, study aimed to establish the previously The photophysical properties of the 152 2þ oxidation state, type of ligands, etc. observed high energy emission along homoleptic complex Ru(bpdz)3 and 3 Thus, the mechanism for light-induced with the time frame of THEXI– MLCT heteroleptic complex [Ru(bpy)2 photo processes in the case of metal formation. Figure 16B shows the Kerr- (bpdz)]2þ [bpdz = 3,30-bipyridazine] 2þ complexes varies widely from each gated TR3 spectra of [Ru(bpy)3] in were studied in detail by Kincaid and 155 other and indeed subtle changes of the acetonitrile (kpump at 400 nm and kprobe co-workers. The TR3 spectrum of the ligand by judicial synthetic chemistry at 350 nm) and water (kpump at 400 nm homoleptic complex contains the ex- can have huge ramifications on the and kprobe at 475 nm ) at different delay pected characteristic frequencies of the photochemical outcomes. times. In the acetonitrile spectra, the bpdz- radical anion. The TR3 spectra Excited-State Behavior of Metal intensity of Raman bands at 1223, 1292, of the heteroleptic complex is dominated Pyridyl Complexes. Polypyridyl ligated and 1547 cm-1, which are signatures of by characteristic bpdz- modes in the metal complexes with d6 (Ru(II), Fe (II), bpy radical anion (bpy–), increases over absence of any modes attributable to and Re(I)) and d10 (Cu (1)) metal atoms the first 20 ps following photoexcitation. bpy- anion radicals, providing evi- used to absorb in the visible region are The probe wavelengths are in resonance dence for identification of the excited- 3 - 2þ attributed to low energy MLCT transi- with the MLCT state, in agreement state species [Ru(III)(bpy)2(bpdz )] . tion and often demonstrate interesting with ultrafast time-resolved absorption The decay pathways of the excited-state photochemical properties that can be spectra. In water the spectra are simpler species formed were found to be differ- used to understand inter- and intra- and the dynamics can be followed by the ent from each other. molecular electron transfer (oxidation 1500 cm-1 band. The rise-time constant The typical family of bpy complexes of metal and reduction of ligand), for THEXI–3MLCT in the above two can be modified by substituting other efficiency, and reliability of photocatal- solvents and also in other homologous ligands such as phenanthroline. Horvath ysis in industrial solar-cell materials.146 alcohols remains similar. Again the rise and co-workers have studied156 a family

1110 Volume 65, Number 10, 2011 2- of metal complexes of [Ru(LL)(CN)4] the ligand allows pp stacking between deuterium), which also acts as a light- type where LL = 1,10-phenanthroline base pairs and results in a decrease in switch reporter.163 Most of the observed (phen) and its methyl- and phenyl- efficiency of the non-radiative relaxation bands are solely attributed to dppz- substituted derivatives and several deu- from MLCT excited state. radical anion (dppz skeleton). terated isotopologues. The objective was The Kerr-gated picosecond TR3 spec- Another ps TR3 report on the light- 2þ to study the difference in optical and troscopic study of the excited states of switch complex [Ru(phen)2dppz] sug- vibrational properties as compared to DNA intercalator system K-[Ru(bi- gests its sensitivity to aqueous and non- 2þ bpy and [Ru(LL)3] complexes. py)2dppz](BF4)2 (where K refers to the aqueous environments and to the pres- For a more detailed understanding of lambda enantiomer) in water and aceto- ence of DNA.164 Here a precursor state the effect of heteroleptic ligand substi- nitrile suggests that the excited-state was identified for the first time on a tution on the excited-state photochemis- structure in both solvents is different shorter time scale in both non-aqueous try, McGarvey and co-workers studied and responsible, at least in part, for their and DNA medium that precedes the two different series of ruthenium(II) photochemical activity.159 The above 3MLCT state and the estimated lifetime polypyridyl complexes, namely complexes are popular for their on–off was observed to be dependent on the þ 160,161 [Ru(bpy)2(phpytr)] and [Ru(bpy)2 luminescence switching, which is environment. A similar light-switch þ (phpztr)] (where phpytr = 2-(5-phe- believed to be related to the excited-state effect was also observed for the complex nyl-1H-[1,2,4]triazol-3-yl)-pyridine and deactivation, which in turn directly formation (covalent attachment or sim- phpztr = 2-(5-phenyl-1H-[1,2,4]triazol- depends on the structure in the excited 2þ ple mixing) between [Ru(phen)2dppz] 3-yl)-pyrazine). It was observed that for state. These molecular systems show and single-stranded (ss) DNA.165 There- the pyridine-1,2,4-triazolato based com- luminescence in water only after inter- fore, it is safe to conclude that the light- plex the lowest excited state is entirely calation with DNA and also exhibit switch effect is not completely depen- bpy based, whereas for the pyrazine- luminescence in less polar solvents such dent on the intercalation between bases. based complexes excited-state localiza- as acetonitrile. It is appropriate to Another analogous system [Ru tion on ligands is dependent on the mention here that any spectroscopic 2þ 157 (tap)2dppz] (tap = 1,4,5,8 tetraazaphe- solvent and pH. In an attempt to observations for study of the ground nanthrene) was also observed to show the understand the effect of the thiophene state using steady-state experiments light-switch effect.166 The nature of the group on the photophysical properties of must be correlated to the excited-state interaction between the probe and DNA triazole-based mononuclear Ru(II) and structure and dynamics using time- depends on the nature and character of Ru(III) polypyridyl complexes, McGar- resolved spectroscopies in different time the lowest excited state. An electron vey and co-workers carried out experi- domains. This was the first ever appli- transfer process between guanine bases ments on Ru complexes based on cation of Kerr-gated ps TR3 towards and the lowest 3MLCT was newly ligands 2-(50-(pyridin-200 -yl)-10H- resolving the structural complexity of an 10,20,40-triaz-30-yl)-thiophene and 2-(50- excited-state inorganic complex.159 In observed for the Ru–tap complex as it (pyrazin-200 -yl)-10H-10,20,40-triaz-30-yl)- water, the electron localized on the intercalates with the DNA. For a finer thiophene.158 Even though the electron- phenazine moiety was found to possess understanding of the light-switch effect, the solvent dependence of various metal withdrawing nature of thiophene groups more MLCT character, which is formed 2þ complexes such as [Ru(bpy)2dppz] , affects the character of the pH-sensitive by electron transfer from the bpy 2þ triazole group, it hardly has an effect on portion. [Ru(phen)2 dppz] ,[Ru(phen)2 2þ the excited-state interaction. The RR spectroscopic investigation of cpdppzOMe] ,andadimer[l- 4þ 2þ 0 C4(cpdppz)2-(phen)4Ru2] was studied [Ru(L)2dppz] (L = 2,2 -bipyridyl calf-thymus DNA intercalation with (bpy); 1,10-phenanthroline (phen)) with dipyridophenazine (dppz) complexes of using Kerr-gated ps TR3 spectrosco- 161 2þ py. The findings of such studies are planar aromatic ligands dppz (dipyrido- ruthenium(II), [Ru(L)2(dppz)] (L = phenazine) is another photochemically 1,10-phenanthroline (1) and 2,2-bipyr- useful in understanding the different interesting system capable of intercala- idyl (2)) was reported by Kelly and co- photoprocesses involving different types tion with DNA. This type of complex workers.162 The 1526 cm-1 Raman of complexes. shows a unique characteristic called the band, which is attributed to the dppz Rhenium Metal Complexes. Rheni- light-switch effect. Briefly, [Ru(L)2 ligand, undergoes changes (reduction to um polypyridyl complexes are consid- dppz]2þ types of systems exhibit very form radical anion) and behaves as a ered as essential photosensitizers for weak 3MLCT luminescence in aqueous marker band for intercalation. Overall, energy and electron transfer reactions solution, which is enhanced in the RR spectroscopy has been tremendously and are an integral part of possible presence of DNA and many non-aque- beneficial for screening metal complexes molecular electronics and photonic de- ous solvents. This signature has been for DNA intercalation interaction simply vices. Another existing practical appli- commonly used in fluorescent probes for due to its ability to follow a higher cation relates to ultrafast electron the detection of DNA. The reason for spectrally resolved Raman marker band. injection from a MLCT excited state in more intense luminescence is ascribed to Similar trends were also observed for the case of solar cells. Therefore, for intercalative binding of the ligands normal and deuterated [Ru(phen)2 better modification and engineering of between the base pairs of DNA. At the dppz]2þ, in which the phenanthroline solar cell materials, a detailed under- same time, the planar aromatic part of hydrogens of dppz are substituted by standing of the ultrafast processes in-

APPLIED SPECTROSCOPY 1111 focal point review volving these d6 types of metal com- tivity than that offered by a single ried out using purpose-designed dye plexes is necessary. technique, thus providing greater confi- lasers and optical components. Howev- A Kerr-gated ps TR3 investigation of dence in the conclusions. er, these provided only a limited number 3MLCT excited states of [Re(Etpy) The photophysical properties of fac- of wavelengths, meaning that the sys- þ þ (CO)3(dmb)] and [Re(Cl)(CO)3(bpy)] [Re(CO)3(dppz)(py)] complex in ace- tems studied had to be carefully chosen (where dmb = 4,40-dimethyl-2,20-bipyr- tonitrile were exploited in the nanosec- to match the available resonance condi- idine) reveals that the intensity of the ond and picosecond domain using a tions. Indeed, sometimes research proj- dominant Raman bands due to vibra- combination of TRA, TR3, and TRIR ects have been selected by their ability tions associated with the NN- ligand spectroscopy.169 The motivation for this to match a set of laser criteria rather than (where NN = dmb, bpy) of the excited study is to understand the variation in academic need. Modern lasers means molecules increases during the first excited-state energy with respect to this is no longer the case. 15-20 ps after excitation. Specifically, ligand substitution, which aids in de- The TR3 technique is highly comple- þ for [Re(Etpy)(CO)3(dmb)] the growth signing molecular inorganic complexes mentary to time-resolved absorption, of Raman bands responsible for intra- with tunable excited states that can be emission, and infrared spectroscopy ligand vibration was estimated to occur used as DNA probes. This is an example and the combinations have been suc- in 6 6 2 ps and is associated with the where time-resolved spectroscopy can cessfully applied for understanding var- growth of signatures correspondng to a open up a scientific arena in which ious complicated reaction pathways. The pp* transition of the dmb- ligand.167 strategic synthesis of complexes can be selectivity of TR3 spectroscopy helps in From the combined TRIR and TR3 exploited to act as a potential DNA probing the structural changes by excit- study of 3MLCT excited states of probe and can be used as other bioinor- ing different chromophores residing in þ [Re(Etpy)(CO)3(dmb)] and [Re(Cl) ganic probes. different pockets of a multi-chromo- (CO)3(bpy)] it is evident that structural In summary, by examining the char- phoric system. This phenomenon is reorganization takes place through vi- acter of a plethora of metal centers and particularly useful for unraveling the brational relaxation/cooling that occurs series of ligand libraries, homoleptic and mechanistic biological interactions in with a time constant of 1–11 ps and heteroleptic metal complexes can be complex systems such as proteins. results in a solvated equilibrium struc- intelligently engineered to provide a Again the high spectrally resolved RR ture that is in accordance with the wide range of ultrafast dynamics, and bands are experimentally proven to be upward shifts and narrowing of anhar- the observations thus obtained can be highly sensitive to change in molecular monically coupled m(CO) IR and dmb- exploited towards practical contributions structure and solution environment. Any Raman bands. These conclusions from in terms of appropriately modifying and change in the structure and environment the ultrafast study are helpful in under- providing working smart materials for is directly reflected in the spectral standing electron injection in semicon- device fabrication affiliated with a wide behavior of the Raman bands. In a ductor materials that occurs from range of sophisticated products. nutshell, TR3 spectroscopy is a sensitive unequilibriated vibrationally excited modality providing structure and reac- states. CONCLUSION AND OUTLOOK tivity information on short-lived com- Another combined time-resolved It has been a goal of the scientific ponents of a photo-reaction in solution spectroscopic (UV-Visible, emission, community to understand and propose phase. IR, TR3) investigation on the complex the mechanisms of light-initiated pro- Significant progress has been made to þ 2þ þ fac-[Re(MQ )(CO)3(dmb)] (MQ = cesses. Time-resolved spectroscopies push the temporal resolution to the N-methyl-4,40-bipyridinium) reveals have proven to be an important tech- femtosecond time domain. Femtosecond that with photoexcitation, the Re-dmb nique and key asset providing a window stimulated Raman spectroscopy 3MLCT state populates and undergoes a for direct observation of reactive inter- (FSRS)24,25,56 and ultrafast Raman loss dmb- ! MQþ inter-ligand electron mediates in real time to support existing spectroscopy (URLS)57–60 are the two transfer (ILET) to form a Re ! MQþ hypotheses as well as stimulating new new weapons that have been added to 3MLCT excited state.168 The time con- avenues of thought on possible reaction the time-resolved spectroscopist’s arse- stants for ILET in various solvents are mechanisms. Time-resolved resonance nal. Both FSRS and URLS constitute a estimated to be in the range of 8–18 ps Raman spectroscopy in the fast and group of potential natural fluorescence and the transition is accompanied by a ultrafast time domains may be consid- rejection techniques that provide back- large structural reorganization of the ered the most valuable technique, with a ground-free, high spectral resolution, MQ and Re(CO)3 moieties. It is very strong track record of promoting our high intensity, neat Raman spectra in clear from the above two examples that understanding of solution-phase photo- the femtosecond time domain (,100 fs). complicated ultrafast dynamics are ex- initiated reactions by resolving the Fluorescence rejection in picosecond pected if the number of types of structure and dynamics of various time-domain spectroscopy has been photochemically active ligands is in- short-lived states and elucidating the achieved by application of a Kerr gate. creased in the heteroleptic metal com- reactivity of all categories of chemical Indeed, the development of Kerr-gated plex. Therefore, it is a trend to employ and biological intermediates from or- fluorescence rejection for ps TR3 spec- complementary time-resolved tech- ganic, inorganic, and biological systems. troscopy and the nonlinear Raman niques in order to provide better objec- The initial TR3 experiments were car- spectroscopies, FSRS and URLS, are

1112 Volume 65, Number 10, 2011 the two major contributions in the 18. S. G. Kruglik, B.-K. Yoo, S. Franzen, M. H. 48. R. Mathies, A. R. Oseroff, and L. Stryer, current decade towards TR3 spectrosco- Vos, J.-L. Martin, and M. Negrerie, Proc. Proc. Natl. Acad. Sci. USA 73, 1 (1976). Natl. Acad. Sci. USA 107, 13678 (2010). 49. G. H. Atkinson and L. R. Dosser, J. Chem. py. In summary, at present TR3 spec- 19. J. Ma, M.-D. Li, D. L. Phillips, and P. Wan, Phys. 72, 2195 (1980). troscopy can be performed on the J. Org. Chem. 76, 3710 (2011). 50. C.-L. Hsieh, M. A. El-Sayed, M. Nicol, M. femtosecond to microsecond time scale 20. R. Anandhi and S. Umapathy, J. Raman Nagumo, and J.-H. Lee, Photochem. Photo- on virtually any sample of interest. The Spectrosc. 31, 331 (2000). biol. 38, 83 (1983). 21. G. Porter, in Fast Reactions and Primary 51. H. Mohapatra and S. Umapathy, J. Phys. technology available today removes Processes in Chemical Kinetics, Proc. 5th Chem. 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1114 Volume 65, Number 10, 2011 Botchway, A. W. Parker, J. G. Vos, W. R. Brady, C. G. Coates, J. G. Vos, and J. J. McGarvey, B. O¨ nfelt, P. Lincoln, B. Norden, Browne, and J. J. McGarvey, J. Phys. Chem. McGarvey, Photochem. Photobiol. Sci. 6, E. Tuite, P. Matousek, and A. W. Parker, J. A 112, 4537 (2008). 386 (2007). Phys. Chem. B 105, 12653 (2001). 152. W. R. Browne, C. G. Coates, C. Brady, P. 158. W. Henry, W. R. Browne, K. L. Ronayne, N. 165.C.G.Coates,J.J.McGarvey,P.L. Matousek, M. Towrie, S. W. Botchway, A. M. O’Boyle, J. G. Vos, and J. J. McGarvey, Callaghan, M. Coletti, and J. G. Hamilton, W. Parker, J. G. Vos, and J. G. McGarvey, J. J. Mol. Struct. 735–736, 123 (2005). J. Phys. Chem. B 105, 730 (2001). Am. Chem. Soc. 125, 1706 (2003). 159. A. C. Benniston, P. Matousek, and A. W. 166. C. G. Coates, P. Callaghan, J. J. McGarvey, 153. P. J. Carroll and L. E. Brus, J. Am. Chem. Parker, J. Raman Spectrosc. 31, 503 (2000). J. M. Kelly, L. Jacquet, and A. Kirsch-De Soc. 109, 7613 (1987). 160. J.-C. Chambron and J.-P. Sauvage, Chem. Mesmaeker, J. Mol. Struct. 598, 15 (2001). 154. R. Horvath, C. A. Otter, K. C. Gordon, A. M. Phys. Lett. 182, 603 (1991). 167. D. J. Liard, M. Busby, P. Matousek, M. Brodie, and E. W. Ainscough, Inorg. Chem. 161. J. Olofsson, B. O¨ nfelt, P. Lincoln, B. Towrie, and A. Vlcek, Jr., J. Phys. Chem. A. 49, 4073 (2010). Norden, P. Matousek, A. W. Parker, and E. 108, 2363 (2004). 155. J. S. Gardner, D. P. Strommen, W. S. Tuite, J. Inorg. Biochem. 91, 286 (2002). 168. D. J. Liard, M. Busby, I. R. Farrell, P. Szulbinski, H. Su, and J. R. Kincaid, J. 162. C. G. Coates, L. Jacquet, J. J. McGarvey, S. Matousek, M. Towrie, and A. Vlcek, Jr., J. Phys. Chem. A. 107, 351 (2003). E. J. Bell, A. H. R. Al-Obaidi, and J. M. Phys. Chem. A. 108, 556 (2004). 156. M. Kova´cs, K. L. Ronayne, W. R. Browne, Kelly, J. Am. Chem. Soc. 119, 7130 (1997). 169. J. Dyer, W. J. Blau, C. G. Coates, C. M. W. Henry, J. G. Vos, J. J. McGarvey, and A. 163. C. G. Coates, P. L. Callaghan, J. J. Creely, J. D. Gavey, M. W. George, D. C. Horva´th, Photochem. Photobiol. Sci. 6, 444 McGarvey, J. M. Kelly, P. E. Kruger, and Grills, S. Hudson, J. M. Kelly, P. Matousek, (2007). M. E. Higgins, J. Raman Spectrosc. 31, 283 J. J. McGarvey, J. McMaster, A. W. Parker, 157. W. R. Browne, W. Henry, P. Passaniti, M. T. (2000). M. Towrie, and J. A. Weinstein, Photochem. Gandolﬁ, R. Ballardini, C. M. O’Connor, C. 164. C. G. Coates, J. Olofsson, M. Coletti, J. J. Photobiol. Sci. 2, 542 (2003).

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