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Raman spectroscopy involves change in the polarizability of the molecular system on excitation and is based on scattering process. Spontaneous Raman scattering is a two photon process, in which the input light initiates the excitation, which then leads to an emission of another photon due to scattering. It is extensively used to understand molecular properties. As spontaneous Raman scattering is a weak process, the detection of these weak Raman photons are rather difficult. Alternatively, resonance Raman (RR) scattering is another technique where the excitation wavelength is chosen according to the material under study. The excitation wavelength is chosen to be within the absorption spectrum of the material under study. RR spectroscopy not only provides considerable improvement in the intensity of the Raman signal, but also provides mode specific information i.e. the modes which are Franck-Condon active in that transition can be observed. There are reports on RR studies of many systems using pulsed light as an excitation source. It is necessary to use at least two pulsed laser sources for carrying out the time resolved RR spectroscopy. A single pulse source for excitation would lead to compromise either with temporal or spectral resolution which is due to the uncertainty principle. If an excitation pulse has pulse width of ~100 femtoseconds then the spectral resolution will be ~ 150 cm-1. It is clear now that for improving the temporal and spectral resolution simultaneously, usage of single pulse for Raman experiments (spontaneous scattering) is not adequate. The usage of multiple laser pulses may provide the way out to improve the resolutions. Nonlinear spectroscopy in a broad view helps in understanding the structural and dynamical properties of the molecular systems in a deeper manner. There are a number of techniques as a part of nonlinear spectroscopy that have emerged in due course to meet different requirements and to overcome some difficulties while understanding the molecular properties. Stimulated Raman (SRS) gain, coherent anti-Stokes Raman scattering (CARS) and the inverse Raman spectroscopy are a few to mention as third order nonlinear spectroscopic techniques which give the similar kind of information about the molecular systems. Stimulated Raman scattering is a more general process involved in nonlinear Raman processes. SRS involves at least two laser pulses and the difference in their frequencies should match with the vibrational frequency of the molecule. The polarization has to be matched between the Raman pump and the Raman probe pulses. We have developed a new nonlinear Raman technique in our laboratory named as ultrafast Raman loss spectroscopy (URLS) using the principles of nonlinear Raman scattering. It involves the Raman pump (~ 1 picosecond (ps) or ~ 15 cm-1spectral resolution) and Raman probe as a white light continuum (100 fs) whose frequency components ranges from 400-900 nm. The laser system consists of Tsunami which is pumped by a Millennia laser and Spitfire-Pro, a regenerative amplifier which is pumped by an Empower laser. Tsunami provides a 100 fs, 780 nm centered, 80 MHz and ~6 nJ energy laser pulses. The Tsunami output is fed into Spitfire to amplify its energy and change the repetition rate to 1 KHz. The pulse length of the input pulse is preserved in amplification. The output of amplifier is split into two equal parts; one part is used to pump the Optical Parametric Amplifier (OPA) in order to generate wavelengths in the range 480-800 nm. The output of the OPA is utilized to generate Raman pump which has to be in ps in order to get the best spectral resolution. A small portion of the other part of amplifier output is utilized to generate white light source for the Raman probe. The remaining part of the amplifier output is used to pump TOPAS to generate wavelengths in the ultraviolet region. URLS has been applied to many molecular systems which range from non-fluorescent to highly fluorescent. URLS has been demonstrated to be very sensitive and useful while dealing with highly fluorescent systems. URLS is a unique technique due to its high sensitivity and the Raman loss signal intensity is at least 1.5-2 times higher as compared to the Raman gain signal intensities. Cresyl violet perchlorate (CVP) is a highly fluorescent system. URLS has been applied to study CVP even at resonance excitation. Rhodamine B has also been studied using URLS. Spontaneous Raman scattering is very difficult to observe experimentally in such high quantum yield fluorescent systems. The variation in the lineshapes of the Raman bands for different RP excitation wavelengths in URLS spectra shows the mode dependent behavior of the absorption spectrum. The experimental observation of variation in the lineshape has been accounted using theoretical formalism. The thesis is focused on discussing the development of the new nonlinear Raman spectroscopic technique URLS in detail and its applicability to molecular systems for better understanding. A theoretical formalism for accounting the uniqueness of URLS among the other nonlinear Raman techniques is developed and discussed in various pictorial representations i.e. ladder, Feynman and closed loop diagrams. A brief overview of nonlinear spectroscopy and nonlinear Raman spectroscopy is presented for demonstrating the difference between the URLS and the other nonlinear Raman techniques.

Tyrosyl radicals can facilitate proton-coupled electron transfer (PCET) reactions that are linked to catalysis in many biological systems. One such protein system is ribonucleotide reductase (RNR). This enzyme is responsible for the conversion of ribonucleotides to deoxyribonucleotides. The beta2 subunit of class Ia RNRs contains a diiron cluster and a stable tyrosyl radical (Y122*). Reduction of ribonucleotides is dependent on reversible, long-distance PCET reactions involving Y122* located 35 Å from the active site. Protein conformational dynamics are postulated to precede diiron cluster assembly and PCET reactions in RNR. Using UV resonance Raman spectroscopy, we identified structural changes to histidine, tyrosine, and tryptophan residues with metal cluster assembly in beta2. With a reaction-induced infrared spectroscopic technique, local amide bond structural changes, which are associated with the reduction of Y122*, were observed. Moreover, infrared spectroscopy of tyrosine-containing pentapeptide model compounds supported the hypothesis that local amide bonds are perturbed with tyrosyl radical formation. These findings demonstrate the importance of the amino acid primary sequence and amide bonds on tyrosyl radical redox changes. We also investigated the function of a unique tyrosine-histidine cross-link, which is found in the active site of cytochrome c oxidase (CcO). Spectrophotometric titrations of model compounds that mimic the cross-link were consistent with a proton transfer role in CcO. Infrared spectroscopic data support the formation of tyrosyl radicals in these model compounds. Collectively, the effect of the local structure and the corresponding protein dynamics involved in tyrosyl radical-mediated PCET reactions are illustrated in this work.

In the first part of this dissertation, recent efforts to understand quantum mechanical effects in biological systems are discussed. Especially, long-lived quantum coherences observed during the electronic energy transfer process in the Fenna-Matthews-Olson complex at physiological condition are studied extensively using theories of open quantum systems. In addition to the usual master equation based approaches, the effect of the protein structure is investigated in atomistic detail through the combined application of quantum chemistry and molecular dynamics simulations. To evaluate the thermalized reduced density matrix, a path-integral Monte Carlo method with a novel importance sampling approach is developed for excitons coupled to an arbitrary phonon bath at a finite temperature. In the second part of the thesis, simulations of molecular systems and applications to vibrational spectra are discussed. First, the quantum dynamics of a molecule is simulated by combining semiclassical initial value representation and density funcitonal theory with analytic derivatives. A computationally-tractable approximation to the sum-of-states formalism of Raman spectra is subsequently discussed.

Ultraviolet-visible spectra are presented for a polycrystalline sample of chlorine clathrate hydrate and two single crystal samples of bromine clathrate hydrate. The data shows that the UV-visible spectroscopy is a sensitive probe for studying the interactions between the halogen guest molecule and the host water lattice. The spectrum for chlorine hydrate shows a surprisingly strong temperature dependence. The spectra reported for bromine clathrate hydrate single crystals reinforce our previous conclusion that there is a stable cubic type II structure as well as the tetragonal structure. There is also a metastable cubic type I structure. The new results are discussed in the context of previous results, resonance Raman spectroscopy, and how the molecules fit into the host cages.

Contemporary laser research involves the development of spectroscopic techniques to understand the microscopic structural aspects of a simple molecular system in chemical and materials to more complex biological systems such as cells. In particular, Raman spectroscopy, which provides bond specific information, has attracted considerable attention. Further with the advent of femtosecond (fs) laser, the recent trend in the field of fs chemistry is to develop nonlinear Raman techniques that allow one to acquire vibrational structural information with both fs temporal resolution as well as good spectral resolution. Among many advanced nonlinear Raman techniques, the development of fs Stimulated Raman scattering (SRS) has gathered momentum in the recent decade due to its ability to (1) provide vibrational structural information of various system including fluorescent molecules with good signal to noise ratio and (2) circumvent the limitation imposed on the spectral resolution by the necessary pulse durations according to the energy-time uncertainty principle where ‘K’ is a constant that depends on the pulse shape) unlike in the case of fs normal resonance Raman spectroscopy. We have developed a technique named “Ultrafast Raman loss spectroscopy (URLS)” that is analogues to SRS, but is more advantageous as compared to SRS and has the potential to be an alternative if not competitive tool as a vibrational structure elucidating technique. The concept and the design of this novel technique, URLS, form the core of the thesis entitled “Ultrafast Raman Loss Spectroscopy (URLS)”. Chapter 1 lays the theoretical groundwork for ultra-short pulses and nonlinear spectroscopy which forms the heart of URLS. It presents a detailed discussion on the basis behind the elementary experimental problems associated with the ultra-short laser pulses when they travel through a medium, the characterization of these ultrashort pulses as well as various non-linear phenomena induced within a medium due to the propagation of these pulses. Chapter 2 focuses on the concept of SRS which resulted into the foundation of URLS. It illustrates the theoretical as well as the experimental aspects of SRS and demonstrates the sensitivity of SRS over normal Raman spectroscopy. Chapter 3 introduces the conceptual and the technical basis which ensued into the development of URLS while Chapter 4 demonstrates its application and efficiency over its analogue SRS. URLS involves the interaction of two laser sources, viz. a picosecond (ps) pulse and a fs white light (WL), with a sample leading to the generation of loss signal on the higher energy (blue) side with respect to the wavelength of the ps pulse unlike the gain signal observed on the lower energy (red) side in SRS. These loss signals are at least 1.5 times more intense than SRS signals. Also, the very prerequisite of the experimental protocol for signal detection to be on the higher energy side by design eliminates the interference from fluorescence, which appears on the red side. Thus, the rapid data acquisition, 100% natural fluorescence rejection and experimental ease ascertain “Ultrafast Raman Loss Spectroscopy (URLS)” as a unique valuable structure determining technique. Further, the effect of resonance on the line shape of the URLS signal has been studied which forms the subject of discussion in Chapter 5. The objective of the study is to verify whether the variation of resonance Raman line shapes in URLS could provide an understanding of the mode specific response on ultrafast excitation. It is found that the URLS signal’s line shape is mode dependent and can provide information similar to Raman excitation profile (REP) in the normal Raman studies. This information can have impact on the study of various dynamical process involving vibrational modes like structural dynamics and coherent control. Chapter 6 demonstrates the application of URLS as a structure elucidating technique for monitoring ultrafast structural and reaction dynamics in both chemical and biological systems using α-terthiophene (3T) as the model system. The objective is to understand the mechanism of the molecular structure dependent electronic relaxation of the first singlet excited state, S1, of α-terthiophene using fs URLS. The URLS data along with the ab-initio calculations indicate that the electronic transition is associated with a structural rearrangement from a non-planar to a planar configuration in the singlet manifold along the ring deformation co-ordinate. The experimental findings suggest that the singlet state decays exponentially with a decay time constant ( 1/e) of about 145 ps and this decay could be assigned to the intersystem crossing (ISC) pathway from the relaxed S1 state to the vibrationally hot triplet state, T1*. Lastly, Chapter 7 summarizes the entire thesis and presents some possible future prospects for URLS. Considering the advantages of URLS, it is proposed that URLS can be exploited [1] to determine the structure of any fluorescent/non-florescent condensed materials and biological systems with a very good spectral resolution (10- 40 cm-1); [2] to obtain the vibrational signature of weak Raman scattering molecules and vibrational modes with relatively small Raman cross-section owing to its high detection sensitivity with good signal to noise ratio; [3] for performing fs time-resolved study by introducing an additional fs pulse for photo-excitation of the molecule and using URLS to probe the excited state dynamics with good temporal (fs) and spectral (10-40 cm-1) resolution; and lastly, [4] the high chemical selectivity of URLS and the fact that the signal is generated only within the focal volume of the lasers where all the beams overlap can be utilized for developing this method into a microscopy for labeled-free effective vibrational study of biological samples. Consequently, it is hoped that this technique, “Ultrafast Raman Loss Spectroscopy (URLS)”, would be a suitable alternative to other nonlinear Raman methods like coherent anti-Stokes Raman spectroscopy (CARS) that has made major inroads into biology, medicine and materials.

Solvent effects play diverse roles in myriads of chemical, physical and biological processes. The solvent interacts with the solute by: i) non-specific (Coulombic, van der Waals interactions) and ii) specific interactions (hydrogen bonding, etc.). These interactions are responsible for solvation of the solute and are collectively termed as “solvent polarity”. Differential solvation of ground and excited electronic states is manifested in the absorption spectrum as a change in the band position, intensity or shape, which is termed as “solvatochromism”. Intermolecular hydrogen bonding (IHB) is a kind of specific solute-solvent interaction, which plays a key role in molecular or supramolecular photochemistry, as well as in photobiology. Solvation and its influence on various physico-chemical and biological processes can be understood by i) top-down; and ii) bottom-up approaches. In the top-down approach, the macroscopic properties like dielectric constant, refractive index are used to understand the microscopic solvation. This approach fails when specific interactions like hydrogen bonding interactions come into play, and furthermore it can reproduce only the macroscopic polarization of the solvent but fails miserably at the cybotactic region of solvation. With the recent advancements in the computational field, the molecular level description of solvation has been within reach for chemical physicists and experimentalists to corroborate their experimental results and in turn to visualize processes of fundamental or technologically relevant problems. The energy levels of the nπ* and ππ* singlet and triplet excited states of aromatic ketones are close-lying and therefore their energy levels can be altered by the substituents. The solvent polarity can be used as a surrogate to tune their energy levels. In certain cases, the lowest triplet or singlet excited states can switch their electronic character with increasing solvent polarity known as “electronic state switching” and thus modulate their photochemical or photophysical properties. Therefore, aromatic ketones were used as solvatochromic probes in this work. Comprehensive analyses of the solvent effects on xanthone (XT), 9,10-phenanthrenequinone (PQ) and benzophenone (Bzp) were carried out using steady-state and nanosecond time-resolved absorption, and resonance Raman spectroscopy in conjunction with ad hoc and classical-molecular dynamics and simulations generated supermolecule-continuum solvent model quantum mechanical calculations to corroborate the experimental outcomes and in turn to visualize the solvation process at the molecular level. The present thesis is divided into eight chapters and the summary of each chapter is described below: Chapter 1 provides a brief literature review of solvation effects and their influence on various physico-chemical and biological processes. Furthermore, the importance of understanding solvation at the molecular level and key concepts are discussed, which forms the heart of this thesis. Chapter 2 discusses the experimental and computational approaches used to study the solvation processes at the molecular level. A detailed explanation of spectroscopic techniques like resonance Raman (RR) and nanosecond-time resolved resonance Raman (ns-TR3) spectroscopy and their experimental and theoretical aspects are discussed, followed by a discussion on the fundamental concepts of computational methods like ab initio calculations density functional theory (DFT), and classical molecular dynamics and simulations (c-MDS) utilized in this study. Chapter 3 focuses on microscopic understanding of solvatochromic shifts observed for 9,10-phenanthrenequinone in protic solvents using UV-Vis and RR spectroscopy in conjunction with an ad hoc explicit solvation model and time-dependent density functional theory (TDDFT) calculations. The hypsochromic shift and bathochromic shift of the singlet nπ* and ππ* electronic transitions in protic solvents are due to hydrogen bond weakening and strengthening in the excited state for the corresponding electronic transitions, respectively as indicated by TD-DFT calculations and Kamlet-Taft linear solvation energy relationships. The hydrogen bond strengthening in the singlet ππ* excited state is further confirmed by Raman excitation profile (REP) analysis of PQ in different solvents. Furthermore, with increasing solvent polarity the two lowest singlet excited states undergo different hydrogen bonding mechanisms, leading to a decreasing energy gap between them. Therefore, hyperchromism of the nπ* transition has been hypothesized to be due to an increasing vibronic coupling between the lowest singlet nπ* and ππ* excited states. In Chapter 4, a real time observation of the thermal equilibrium between the lowest triplet excited states of PQ in acetonitrile solvent was carried out using ns-TR3 spectroscopy and this can explain its high reactivity towards H-atom abstraction, despite the fact that the lowest triplet excited state has ππ* character. Furthermore, extending the concept of hydrogen bonding mechanisms of the lowest singlet to the triplet excited states, the different hydrogen bonding mechanisms exhibited by them can lead to alteration of the intersystem crossing mechanisms in PQ. Chapter 5 highlights the very different role of intermolecular hydrogen bonding in the reduced reactivity of the xanthone (XT) triplet towards H-atom abstraction in protic solvents. The different hydrogen bonding mechanisms exhibited by the two lowest triplet excited states in protic solvents are derived from an ad hoc explicit solvation model, TD-DFT calculations and ns-time resolved absorption (ns-TRA): they separate them further in energy and thereby the nearest T2(nπ*) triplet state to the T1(ππ*) excited state plays an insignificant role in the reactivity towards H-atom abstraction, in contrast to the PQ triplet discussed in Chapter 4. Chapter 6 discusses the structure of XT triplet states using TR3 spectroscopy in combination with TD-DFT studies. The TR3 spectrum of the XT in acetonitrile identified a vibronic coupling mode responsible for the reactivity of XT towards H-atom abstraction, despite the fact that the lowest triplet excited state (T1) has ππ* character. This vibronic active mode is absent in the TR3 spectra of XT in protic solvents (methanol and ethanol). Furthermore, the REP analysis suggests that the nanosecond triplet-triplet absorption spectrum of XT in acetonitrile involves two different species, while in methanol it involves only one species. This observation is in agreement with the previous chapter (Chapter 5) which proposes a different hydrogen bonding mechanisms for the two lowest triplet excited states and their influence on the reduced reactivity towards H-atom abstraction. Chapters 3-6 emphasize the need for a proper solvation model at the molecular level to describe the various photophysical and photochemical processes of aromatic ketones. Therefore, Chapter 7 includes discussions on the bottom-up solvation methodology applied to benzophenone (Bzp) to understand its vibrational and electronic solvatochromic behaviour at the molecular level. Raman and UV-Vis spectroscopic techniques were used in conjunction with a c-MDS-generated supermolecule continuum solvation model DFT calculation to corroborate and to visualize the experimental outcome. The carbonyl stretching frequency of Bzp in protic solvents has two bands, corresponding to free carbonyl and hydrogen bonded carbonyl. Despite the fact that the macroscopic polarity of acetonitrile and methanol solvents are similar, the free carbonyl stretching of Bzp in methanol solvent was blue-shifted by 4 cm-1 with respect to the carbonyl stretching in acetonitrile solvent. The Gutmann’s acceptor number plot for carbonyl stretching frequencies indicates that the free carbonyl group is neighboured by a hydrophobic environment. The c-MDS-generated supermolecule-continuum solvation model DFT calculations suggest that the extended hydrogen bonding network of methanol solvent is responsible for the hydrophobic solvation around the free carbonyl. Furthermore, a linear correlation was obtained for the vibrational and electronic solvatochromism of the carbonyl frequency and energy of the singlet nπ* transition, respectively, which indicates that a variation in excitation wavelength for the singlet nπ* transition can arise from different solvation states. This can have implications for ultrafast processes associated with electron transfer, charge-transfer and also the photophysical aspects of excited states.Finally, Chapter 8 contains overall conclusions of the thesis and future directions for the present research area.

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