A COMBINED EXPERIMENTAL AND THEORETICAL INVESTIGATION ON THE SURFACE ENHANCED RAMAN SCATTERING OF SOME ORGANIC MOLECULES ADSORBED IN METAL HYDROSOL

Transcription

1 A COMBINED EXPERIMENTAL AND THEORETICAL INVESTIGATION ON THE SURFACE ENHANCED RAMAN SCATTERING OF SOME ORGANIC MOLECULES ADSORBED IN METAL HYDROSOL THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (SCIENCE) OF JADAVPUR UNIVERSITY 2008 By JYOTIRMOY SARKAR, M.Sc. DEPARTMENT OF SPECTROSCOPY INDIAN ASSOCIATION FOR THE CULTIVATION OF SCIENCE KOLKATA: INDIA

2 INDIAN ASSOCIATION FOR THE CULTIVATION OF SCIENCE Jadavpur, Calcutta , INDIA From: Prof. G. B. Talapatra, Senior Professor, Dept. of Spectroscopy. Certificate This is to certify that the thesis entitled "A Combined experimental and theoretical investigation on the Surface Enhanced Raman Scattering of some organic molecules adsorbed in metal hydrosol" submitted by Sri Jyotirmoy Sarkar who got his name registered on 14 November, 2006 for the award of Ph. D. (Science) degree of Jadavpur University. The work described in this thesis is absolutely based upon his own work carried under my supervision and that neither this thesis nor any part of it has been submitted for any degree/diploma or any other academic award any where before. (G. B. TALAPATRA) Signature of Supervisor & Date with official seal. Phone: Fax:

3 Synopsis The thesis entitled "A Combined experimental and theoretical investigation on the Surface Enhanced Raman scattering of some organic molecules adsorbed in metal hydrosol" comprises of eight chapters. This focuses on the present dissertation is to investigate the structure, orientation, conformation, binding mechanism and reactivity of some organic molecules adsorbed on nano colloidal silver surface using FTIR, NRS and SERS technique. The contents of the thesis are given below. Chapter 1 contains the general introduction with a brief review of the relevant parts of existing theories employed in this work to interpret the experimental results. Chapter 2 discusses the methods of sample preparation, purification and various experimental techniques of NRS, SERS, and FTIR. Chapter 3 deals with the detail experimental and theoretical NRS, SERS and FTIR spectra along with a tentative vibrational assignment of the observed bands of the biologically important, 2-aminobenzothiazole (2-ABT) molecule. The optimized structural parameters and the computed vibrational wavenumbers of the compound have been estimated from ab initio HF and DFT calculations. Some vibrational modes of the molecule have been reassigned. The adsorptive behavior of 2-ABT on a colloidal silver surface at different adsorbate concentrations, close to that encountered under physiological conditions in living systems has been elucidated from the SERS spectra. NRS spectra of the chemically prepared 2-ABT-Ag(I) complex and their comparison with the SERS spectra are also reported herein. Chapter 4 presents the detail experimental and theoretical NRS, SERS and FTIR spectra along with a tentative vibrational assignment of the observed bands of the 2-Amino-4-Methyl Benzothiazole (2-AMBT). The adsorptive behavior of these molecules on the colloidal silver surface at two different adsorbate concentrations recorded in different time domains have been elucidated from the SERS spectra. The silver surface may serve as an analogue for the artificial biological interface. The experimentally observed SERS spectra are compared with the theoretically modeled 2-AMBT-Ag (I) surface complexes using ab initio RHF and DFT calculations. The

4 most favorable adsorptive sites of the 2-AMBT molecule have been estimated by natural population analysis (NPA) using the above-mentioned high level of theories. Chapter 5 discusses the studies on the experimental and theoretical NRS, SERS and FTIR of 2-Amino-6-methylbenzothiazole (2A-6MBT) molecule. The adsorptive behavior of 2A-6MBT molecules on the colloidal silver surface at different adsorbate concentrations has been elucidated from the SERS spectra. In these investigations, the silver surface may serve as an analogue for artificial biological interface, and after elucidating the adsorption mechanism of the molecule; the study can be extended to the adsorption on membranes or other interesting biological surface for medical or therapeutic treatments. DFT calculations on models of 2A- 6MBT-Ag 0 and 2A-6MBT-Ag + surface complexes are also reported herein. Chapter 6 reports the adsorptive behavior of Rh123 on the colloidal silver surface and the nature of charge transfer between the molecule and the metal using FTIR and SERRS spectra together with ab initio and DFT calculations. This study may be helpful to understand the role of this molecule at biological interfaces. Chapter 7 deals with the detailed experimental and theoretical normal Raman spectra (NRS), SERS and FTIR spectra of 4-Methyl-4H-1, 2, 4-Triazole-3-Thiol (4- MTTL) molecule. From a more fundamental point of view, 4-MTTL is also very interesting compound because of its probable existance in thione-thiol tautomeric equilibrium in electronic ground state. The ph dependent NRS spectra of the molecule in aqueous solution have been recorded to elucidate the protonation effect and preferential exitence of the tautomeric form/forms of the molecule in acid, neutral and alkaline media. The adsorptive behavior and the orientation of the molecule on the nanocolloidal silver surface at various ph values are also recorded herein. Chapter 8 reports the results of the investigation on the concentrationdependent SERS study of the biologically important, 2ATH molecule, adsorbed on silver nanocolloids is described and compared with its FTIR and NRS spectra in varied environments. The optimized structural parameters, preferential existence and computed vibrational frequencies of the tautomeric amino and the isomeric imino forms of the molecule in the gas phase and in methanol solvent have been estimated from the DFT calculations. The observed Raman signals along with the corresponding FTIR bands have been assigned and presented for the first time from the potential energy distributions (PED) in terms of internal coordinates of the molecule estimated from the results of DFT calculations. The adsorptive behavior and adsorption

5 geometry of the preferred tautomeric form/forms of the molecule on nanocolloidal silver surface at different adsorbate concentrations, close to that encountered under physiological conditions in living systems have been elucidated from the SERS spectra.

6 Acknowledgement Research is a task requiring patience, perseverance and strength of mind. There are moments of utmost joy as well as of great disappointment. I have been fortunate enough in having the able guidance and constant encouragement from my supervisor Professor G. B. Talapatra throughout the course of my research work. I would like to express my deep sense of gratitude and indebtedness to Dr. Joydeep Chowdhury for his valuable help, suggestions and effort with day-to-day experiments that has culminated in various publications. His understanding attitude and spontaneous compassion, in the face of experimental reverses, have made long periods of diligent work, a pleasure. I am very much grateful to Dr. Rina de, Dr. Manash Ghosh, Dr. Prabir Pal for their constant guidance and encouragement particularly during my first step into research career. It was indeed been a pleasure working with them. I would like to thank my colleagues Santanu, Narayan, Bishu and my junior Tapanendu for their friendly and academic companionship. Special thanks are also due to Mrs. Sanhita Podder, my wife, for her constant moral support and inspiration during the preparation of this thesis. I am also grateful to the authority of the Indian Association for the Cultivation of Science for their award of a fellowship and providing excellent laboratory, workshop and library facilities. Last but not the least, I must express my deepest gratitude to my parents for their cooperation and help regarding family matters which enabled me to devote utmost energy for the successful completion of my thesis work. Department of Spectroscopy, I.A.C.S., Kolkata November, 2008 (JYOTIRMOY SARKAR)

7 Contents Chapter Introductory Remarks Aim of the thesis General Introduction The Raman Effect and Normal Raman Scattering The Scattering Process Mechanism involved in the phenomenon of SERS 08 a) Classical Electromagnetic Mechanism (i) Isolated metal particles 09 (ii) Collective Resonances. 14 (iii) Quadrupole polarizability mechanism 16 (iv) Molecule in a trap: Multipolar treatment (v) SERS on Flat surfaces: Image field theory.. 19 (vi) Recent Development on the electromagnetic mechanism of SERS 20 (vii) Inadequacy of the electromagnetic model in explaining the overall SERS phenomenon. 21 b) Charge-transfer mechanism of SERS.. 23 (i) Ground state charge transfer.. 23 (ii) Excited state charge transfer. 23 (iii) Recent Reports on the Charge Transfer Mechanism SERS selection rule SERS of Molecules adsorbed in Langmuir-Blodgett (LB) Film Fibre-Optical SER Tip Enhanced Raman Scattering (TERS) Single Molecule Detection Using SERS Quantum Chemical Calculations: Ab Initio and Density Functional Theory 33 Methods a) Ab initio Method.. 33 b) Density Functional Theory Method Normal Coordinate Analysis. 38 (i) Internal coordinates 39 (ii) Equation of motion 39 (iii) Vibrational normal modes and potential energy distribution Two-Dimensional Correlation Spectroscopy Applications of SERS in areas of contemporary interest Limitations of the SERS technique Reference.. 45 Chapter Introductory Remarks Preparation of Silver Nanocolloid Sample Preparation and Purification Measurement of Absorption Spectra.. 57

8 2.5 Fourier Transform Infrared Absorption Measurement Raman Spectra Measurement Spectra Physics Model Ar+ Laser Sample Chamber (Illuminator) Spex Double Monochromator Model Photomultiplier Tube Data Acquisition System Computational Software. 63 Reference.. 64 Chapter Introductory Remarks Results and Discussion Normal Raman and FTIR spectra of 2-ABT Concentration-Dependent SERS spectra of 2-ABT Orientation of the 2-ABT Molecule on the Silver Surface NRS of the 2-ABT-Ag Complex Conclusion.. 80 Reference.. 81 Chapter Introductory Remarks Results and Discussion Normal Raman and FTIR spectra of 2-AMBT and their vibrational 84 assignment SERS Spectra of 2-AMBT Orientation of the 2-AMBT Molecule on the Silver Surface DFT and ab Initio Calculations on Models of Surface Complexes Electronic Absorption Spectra of Silver Colloid with Added 2-AMBT Excitation Wavelength Dependence Conclusion Reference Chapter Introductory Remarks Results & discussion Normal Raman and FTIR spectra of 2A-6MBT and their vibrational 105 assignment Concentration-dependent SERS spectra of 2A-6MBT Orientation of the 2A-6MBT molecule on the colloidal silver surface DFT Calculations on models of surface complexes Direction of CT mechanism in the SERS of 2A-6MBT molecule Conclusion Reference.. 123

9 Chapter Introductory Remarks Results & Discussion Molecular Geometry of Rh Normal Raman, FTIR spectra and vibrational analysis of Rh SERRS spectra of Rh Electronic absorption spectra of silver colloid with added Rh Conclusion Reference Chapter Introductory Remarks Results & Discussion Molecular structure Normal Raman and FTIR Spectra of the molecule and their 141 Vibrational Assignment NRS and FTIR spectrum of the molecule in solid state ph dependent NRS spectra of the molecule in aqueous solution ph dependent SERS Spectra of the molecule Orientation of the molecule on the silver surface Conclusion References. 162 Chapter Introductory remarks Results & Discussion Molecular structure Normal Raman and FTIR Spectra of the molecule and their 168 Vibrational Assignment Concentration-Dependent SERS spectra of the molecule Orientation of the molecule on the colloidal silver surface Conclusion References. 189 APPENDIX: LIST OF PUBLICATIONS List of research papers published Paper presented in National / International Conferences / Symposium 192

10 1 CHAPTER 1 Introduction 1.1 Introductory Remarks Vibrational spectroscopy of molecules adsorbed on metal surfaces is extremely effective in the detection of the adsorption-induced changes in a molecule. Infrared transmission/absorption or reflection spectroscopy, electron tunneling spectroscopy etc. are the widely used surface sensitive technique. Surface enhanced Raman scattering (SERS) is a recent addition to these spectroscopic techniques, which has developed into a diagnostic probe for the analytical characterization of the adsorbates and microscopic structure of surfaces and interfaces. Normal Raman scattering (NRS), however is a weak process, characterized by cross sections of ~10-29 cm 2. Moreover, the NRS is often interfered with fluorescence emission. Thus, it is not suitable for vibrational studies at low concentration of the sample. However, the discovery of SERS has changed the situation and Raman signals are now distinctly detectable at trace concentrations down to single molecule detection level. It is a useful tool in surface chemistry and physics because of its high sensitivity and potential in providing useful information regarding metal-adsorbate interactions. Recent advances have made SERS a versatile technique having a diverse field of application, not only in analytical science but also in biomedicine, environmental monitoring, and artwork conservation and in nanotechnology. Although the origin of SERS remains a matter of controversy, it is now widely accepted that there are two main contributions to the overall effect. One is the classical electromagnetic contribution through the surface plasmon resonance by the exciting electromagnetic field and the other is the chemisorption, which involves charge transfer (CT) interaction between the adsorbed molecule and the metal surface. 1.2 Aim of the thesis The primary aim of the present dissertation is to investigate the structure, orientation, conformation, binding mechanism and reactivity of some organic

11 2 molecules adsorbed on nano colloidal silver surface using FTIR, NRS and SERS technique. Such studies have been done on benzothiazole and its derivatives those are widely recognized as corrosion inhibitors and have been extensively used in the surface treatment of metals and alloys. Moreover, benzothiazoles also form an important class of chemical species, which are involved in numerous applications, including human and veterinary medicine. Detailed vibrational analyses with the aid of ab initio and Density functional theoretical (DFT) calculations have been performed to have a precise idea of vibrational assignment and tautomeric preference of the molecules. SERS of some biologically and industrially significant organic molecules have been extensively studied to understand the adsorption mechanism, adsorptive site/sites and their respective orientations on colloidal silver particles through detailed analysis of the spectral patterns and intensity enhancements of vibrational bands. The theoretical calculations based on the ab initio and DFT were performed using Gaussian software. The combined theoretical and experimental results give us an idea about the orientation; adsorptive site/sites of the molecules adsorbed on colloidal silver surface and help us to apprehend the direction of charge transfer (CT) mechanism of SERS. Generalized 2D-correlation spectroscopic technique has also been successfully utilized to understand and envisage the preferential nature of the adsorbed species of tautomeric 4-Methyl-4H-1,2,4-Triazole-3-thiol and 2-Amino-2- Thiazoline molecules on the nanocolloidal surface. This chapter outlines the relevant things needed to understand the results of the work. 1.3 General Introduction In the Raman Effect, electromagnetic radiation is inelastically scattered from a sample and shifted in frequency by the energy of its characteristic molecular vibrations. It probes vibrational levels of the molecule, which depend on the kinds of atom and their bond strengths and arrangement in a specific molecule. Therefore, a Raman spectrum provides a structural fingerprint of a molecule. Although it is a powerful spectroscopic technique with many practical applications, the main disadvantage of Raman spectroscopy is that it has extremely small scattering cross sections, orders of magnitude below the fluorescence cross section. The advent of laser sources having monochromatic photons at high flux densities is a milestone in

12 3 the history of Raman spectroscopy and resulted in dramatically improved scattering signals. The study of chemical and physical process at interfaces is fundamental not only to the surface sciences, but also to many other branches of fundamental and applied sciences. An important challenge that facing the analytical chemistry is not only detecting trace amounts of substances, but also identifying their chemical structure, for example by Raman scattering. In 1974, about 50 years after the discovery of the Raman Effect, Fleischmann and coworkers [1] discovered the novel phenomenon the SERS. They reported an extraordinary million-fold enhancement of Raman signal from pyridine molecules adsorbed (from solution) onto electrochemically roughened silver electrode compared to that from free molecules in liquid environment. This enormous enhancement of Raman scattering cross section from molecules adsorbed to metallic nanostructures attracted considerable significance from both basic and practical viewpoints. After Fleischmann in year 1976, Richard Van Duyne and David Jeanmaire at Northwestern University observed the effect [2] and in early 1977 M. G. Albrecht and J. A. Creighton reported similar observation [3]. This surprising discovery touched off a flurry of theoretical and experimental activity. Despite all the major theoretical investigations related to the mechanism of SERS, there is still much debate about the exact mechanisms responsible for this effect. However, the mechanism of SERS enhancement remains an active research topic. As mentioned earlier, to date, although, the theoretical understanding of SERS is not clear, but it is accepted that two mechanisms have been shown to be contributing to this enormous enhancement of Raman scattering: One is chemical enhancement and the other one is electromagnetic enhancement. In the chemical mechanism, a charge transfer state is created between the metal and adsorbate molecules due to mixing of the orbital of the adsorbed molecule and the metal atoms, which is now roughly to contribute an average enhancement factor of 100 [4]. Direct involvement between the adsorbate molecules and the roughened metal surface is required to experience the chemical enhancement. On the other hand, the electromagnetic mechanism contributes an average enhancement factor of greater than 10000, which arises from the enhancement of the local electric fields, in the proximity of metal surface, due to the excitation of surface plasmon modes and the

13 4 modulation of metallic reflectance due to adsorbate vibrations. This model does not require specific bonds between the adsorbates and metals. Figure 1.1 shows a cartoon of electric field localization in colloids and sharp point samples. The field intensity depends on the inter-particle distance and particle shape Figure 1.2 shows pictorially the SERS process steps. Figure 1.1 A cartoon of electric field localization in colloids and sharp point samples. Figure 1.2 SERS process steps: (1) laser light incident on the metal substrate (2) plasmons excitation (3) light scattered by the molecule (4) Raman scattered light transferred back to plasmons and (5) scattered in air The surface plasmon can be viewed as collective charge oscillation at the metal air interface. Surface plasmons either can be propagating on the surface of the metal or localized on the surface of a spherical particle. Surface roughness or curvature is required for the excitation of surface plasmons by light. Plasmons at the metal act as antennas, which assist in coupling light into molecules close to the surface and couple out photons into specific directions. This coupling both into and out of the molecule enhances the Raman signal. Thus, the electromagnetic field of the light at the surface can be greatly enhanced under conditions of surface plasmon excitation; the amplification of both the incident laser field and the scattered Raman field through their interaction with the surface constitutes the electromagnetic SERS mechanism. There have been many versions of the electromagnetic theory developed over the years that treat physical situations of varying complexity at different levels of completeness. Model systems that have been treated include isolated spheres, isolated ellipsoids, interacting spheres and ellipsoids, randomly rough surfaces treated as

14 5 collections of hemispherical bumps or gratings and fractal surfaces. These systems have been analyzed with different degrees of sophistication. The simplest treatments invoke the electrostatic approximation using sharp boundaries and local, bulk dielectric functions for the substrate. The discovery of SERS has produced significant impact in fields such as surface chemistry, solid-state physics, inorganic chemistry of metals, electrochemistry because of its high sensitivity and potential in providing useful information regarding metal-adsorbate interactions. The plasmon properties such as wavelength and width of its resonance depend on the nature of the metal surface and on its geometry. Many early SERS substrates used a random roughening of the surface so only small, uncontrolled areas of the total metal surface would have the correct geometry for Raman enhancement. Most traditional techniques of preparing SERS surfaces have therefore been plagued by 100% variations in the Raman signal across the surface and by hot spots where only small areas of the total devices had the right metal geometry for SERS enhancement. Predominantly SERS phenomenon exhibits only a few metals such as silver, gold and copper and these metals are considered efficient SERS active surfaces. However, other metals like Rh [5], Ga [6] and Pt [7] have also been claimed to exhibit enhancement. Very recently, rhenium oxide (ReO 3 ) nanocrystals used as a significant SERS substrate by Rao et al [8]. SERS are also observed at metal-gas interfaces [9], solid-solid interfaces [10], metal sols of silver, gold and copper [11, 12], metal island films [13], chemically deposited silver films and acid-etched silver foils [14]. Gliemann et al [15] reported that commercially available photographic paper for black and white prints could be used successfully to generate attractive SERS-active surface. Apart from various substrates used for SERS studies as we discussed earlier, now some transition metals are also used as a SERS substrates. Tian et al [16, 17] were the first obtained a good quality of SERS spectra from bare transition metal electrodes. Recently some spherical core shell metal nanoparticles like Gold core palladium shell [18], silver core gold shell [19] and not only spherical metals particles but also different size and shape like nanobar, nanorice of silver [20], nanowires, nanoplates of silver [21] and gold nanoparticles of shapes like cubes, blocks,

15 6 tetrapods, spheres and rods [22] are shows a beautiful SERS signal. Metal nanorod and nanowire arrays are now successfully utilized as new and well-structured SERS substrates [23-25] The Raman Effect and Normal Raman Scattering As mentioned earlier, when light is scattered from a molecule most photons are elastically scattered. The scattered photons have the same energy as the incident photons. However, a small fraction of light (approximately 1 in 10 7 photons) is scattered at optical frequencies different from, and usually lower than, the frequency of the incident photons. The process leading to this inelastic scattering is termed as the Raman Effect. Raman scattering can occur with a change in vibrational, rotational or electronic energy of a molecule. C. V. Raman and Krishnan observed this phenomenon in the 1920 when monochromatic light passes through a liquid [26]. The difference in energy between the incident photon and the Raman scattered photon is equal to the energy of a vibration of the scattering molecule. A plot of intensity of scattered light versus energy difference is a Raman spectrum The Scattering Process Figure 1.3 shows quantum mechanical energy level diagram of Rayleigh and Raman scattering. Figure 1.3 Quantum mechanical Energy level diagrams of Rayleigh and Raman scattering. The vibrational states show the difference in emission photons associated with Rayleigh scattering and Raman scattering. (a) Stokes Raman scattering (b) Anti-Stokes Raman scattering

16 7 The first arrow shows the energy contained in the incident photon, which strikes the molecule. The molecule is excited to a virtual excitation state and not a fully excited state. This occurs in both Rayleigh and Raman scattering. In Rayleigh scattering, a photon is released with the same amount of energy as in the incident photon. If the incident photon of frequency ν strikes the molecule then the total c energy in the incident photon is Etotal = hν = h, where h is Planck s constant, c is λ the speed of light, and λ is the wavelength in centimeters. This is also called elastic scattering. Raman scattering is indicated in Figure 1.3 by the second emission arrow. The decrease in energy is smaller than in Rayleigh scattering. In Raman (inelastic) scattering, the exciting photon leaves the molecule with less energy than the incident photon. The energy, due to the inelastic scattering, is lost for the vibrations of the molecular bonds after the transfer of energy to the molecule: E = hν where E is the difference in energy and ν vib is the vibrational frequency of the molecular bond that is vibrating. The Raman scattered photon has less energy and is at a longer wavelength as the total energy of the incident photon must be conserved. Monitoring these inelastically scattered photons will give the information about the vibrations occurring in the molecule, which is nothing but a Raman spectrum [27, 28] where E = h ( ν ν ) (1.1) Raman In other words, the Raman Effect arises when a photon is incident on a molecule and interacts with the electric dipole of the molecule. It is a form of electronic (more accurately, vibronic) spectroscopy, although the spectrum contains vibrational frequencies. In classical terms, the interaction can be viewed as a perturbation of the molecule s electric field. In quantum mechanics, the scattering is described as an excitation to a virtual state lower in energy than a real electronic transition with nearly coincident de-excitation and a change in vibrational energy. The scattering event occurs in seconds or less. The virtual state description of scattering is shown in Figure 1.3a. At room temperature, the thermal population of vibrational excited states is low, although not zero. Therefore, the initial state is the ground state, and the scattered photon will have lower energy (longer wavelength) than the exciting photon. This vib vib

17 8 Stokes shifted scatter is what is usually observed in Raman spectroscopy. Figure 1.3a depicts Raman Stokes scattering. A small fraction of the molecules is in vibrationally excited states. Raman scattering from vibrationally excited molecules leaves the molecule in the ground state. The scattered photon appears at higher energy, as shown in Figure 1.3b. This anti-stokes-shifted Raman spectrum is always weaker than the Stokes-shifted spectrum, but at room temperature, it is strong enough to be useful for vibrational frequencies less than about 1500 cm -1. The Stokes and anti-stokes spectra contain the same frequency information. The ratio of anti-stokes to Stokes intensity at any vibrational frequency is a measure of temperature. Raman spectroscopy is complementary to another vibrational spectroscopy method, infrared spectroscopy. Infrared spectroscopy also gives bond vibration information but it is based on the absorption of infrared light by the molecule. The peaks in each of the forms of spectroscopy describe similar vibrational bands in a spectrum and occur in comparable locations within the spectrum [27] Mechanism involved in the phenomenon of SERS More than 20 years after the first observation of enhanced Raman spectra of pyridine, the method has now reached an advanced state of development yet the exact mechanism of SERS is still under controversy. As Raman scattering arises from the induced dipole moment P produced by the interaction of electromagnetic field of light E with the molecular polarizabilityα, mathematically connected as P = α E, where P and E are vectors and α is a tensor of rank 2. So the origin of tremendous enhancement in Raman scattering is obviously due to any type of perturbation in either one or both of E and α. Based on this fundamental concept, it is now unanimously accepted that two simultaneously operative mechanisms, a long-range classical electromagnetic effect (EM), which involves perturbation in E and a short-range chemical effect (CHEM) which involves perturbation of polarizability,α are generally accepted to be responsible for SERS. a) Classical Electromagnetic Mechanism Electromagnetic theory relates the optical properties of materials to their electrical conductivities. Transparent substances are poor electrical conductors.

18 9 Metals have high conductivity and practically opaque to photons of low energy. The strong absorption in metals is also accompanied by high reflectivity. The optical properties of molecules at metal surfaces are determined in part by the electric field associated with the reflected light. A beam of light reflected from metal surface at normal incidence produces a standing wave electric field, which has a node close to the surface of the metal. However, for a particular, generally high angle of incidence, the incident and the reflected electric vectors combine to give a resulting standing wave field with significant amplitude at the surface, largely normal to the surface. Metals are plasmas where mobile charges are conduction electrons, which for some metals are relatively free to move through the volume of the metal behaving as a free electron gas. The electron gas in metal can undergo collective longitudinal oscillations. The displacement of electron gas as a whole with respect to the positive ion background creates a coulombic restoring force and plasma oscillations result. A plasmon is a quantum of energy of such plasma oscillations. At the surface of the metal, where electronic properties differ from those in the bulk, an additional mode of plasma oscillation exists when the sum of the relative permittivity of the metal and the medium is zero. These surface plasma oscillations, sometimes called surface electromagnetic waves, propagate parallel to the surface. The dispersion relations of surface plasmons result in two types of modes: radiative surface plasmons and non-radiative surface plasmons. These are distinguished by their ability to couple directly to the electromagnetic field. The latter are localized in a region about the metal surface and are of primary importance in metal surface studies. These decay exponentially with the distance from the interface. The resonant excitation of surface plasmons by electromagnetic radiation gives rise to large local fields and this has important consequences in field-dependent phenomenon. (i) Isolated metal particles It is very difficult to prepare an ensemble of non-interacting particles, having either, a known random, or a known periodic distribution, atomically smooth surfaces of identical sizes and shapes for accurate electromagnetic calculations. Carefully prepared colloidal metal particles or metal particles trapped in solid matrix are the most consonant systems that agree approximately with the metal particle model. Another similar system can be prepared by condensing metal vapor on the top of a

19 10 periodic array of vertical pillars made on a photoresist surface. By spacing the pillars properly, the magnitude of the electromagnetic interactions between the metal particles can be controlled. Metiu and Das [29] described the local field enhancement for the case of a sphere of diameter smaller than the wavelength of light. A dipole moment P( ω) given by induced at the center of the sphere by the incident field E0( ω) is P ( ω)= βω ( ) E ( ω) = [ εω ( ) ε][ εω ( ) + 2 ε] a E ( ω) Where ε( ω)and a are the dielectric constant and the radius of the sphere (1.2) ε ( ω ) 0 is the dielectric constant of the surrounding medium, β( ω)represents the polarization of the sphere. The induced dipole creates a field ( r, ω ) at the molecular location of r 0 Ε 0 0 (the centre of the sphere being the origin of the co-ordinate system) [30] and is given by E r ω r P ω r r P P r rr I r P ω 3 3 (, ) ( ). ( ) [ 3 ˆ( ˆ. ) ] =I = = ( 3 ˆˆ ). ( ) Where r0 r0 r = 0 and ˆ r is a dyadic [i. e., r r r ˆ. P r ˆ ( r ˆ. P) 0 ˆ0 ˆ = ] (1.3) Now, the incident electromagnetic field, say, Ε ( r, t) = Ε ( r, ω ) e iω t, acting 0 0 on the molecule is increased through the addition of a field caused by the polarization of the surface. A molecule located at r 0, is thus exposed to the local electric field, which is the incident field that is present at the molecular position ( r 0 ) when the molecule is absent and the other is the field caused by radiation emitted by induced dipole at some past time and reflected back to position r 0 by the surface. Thus, Efrima and Metiu [31-32] express the field at E Where the reflected field laser and I r 0 as ( r ω ) [ Ι + R( r, ω)]. Ε ( r, ) 0, ω = (1.4) R.E 0 is caused by the polarization of the surface by the is a unit tensor. Enhancement occurs because E >> E0. The reflection tensor R is obtained by combining equations (1.2), (1.3) and (1.4) as

20 R ( r 0, ω ) = β( ω ) I( r 0 ) (1.5) The reflection tensor R 11 is the shorthand notation indicating that by solving Maxwell s equations we can express the field caused by surface polarization in terms E of the incident field and the properties of the surface (contained in R ). In case of 0 perfectly conducting, planar-surface, the effect of R amounts to putting a mirror behind the molecule, to increase the amount of light incident on it. The reflection tensor R has an electromagnetic resonance, which minimizes the denominator in equation (1.3) at frequency ω r given by Re ε( ω r ) = 2ε 0 (1.6) A small sphere is capable of sustaining an infinite number of resonances [33] located at the frequencies ω r given by Re ε ( ω r ) = [( n+ 1) / n] ε 0 (1.7) Where n = 123,,... etc. The resonance displayed by the reflection tensor corresponds to n = 1 in equation (1.7). The absence of other resonances in R has been explained by Metiu and Das [29] by concerning the incident field is spatially smooth over the region occupied by the sphere and therefore can excite only the dipolar resonance n = 1 and the excitation of the resonances n > 1are radiation less transitions. The molecular dipole µω ( / ) transfers energy to the resonance n = 1, which / represents enhanced emission. The molecular dipole exerts the field I ( ). µω ( ) on the sphere inducing the radiating dipole ( ω ) = β ( ω ) I. µ ( ω ) P (1.8) From equations (1.5) and (1.8) it is expected that for the excitation frequency close to the emission frequency (i.e. for ω ω / < the width of the electromagnetic resonance) the two processes are equally effective. In Raman scattering ω ω / r 0 is the vibrational frequency. Thus, the high frequency Raman modes are expected to be enhanced less

21 12 than the low frequency modes because in the later case simultaneous resonant enhancement of both excitation and emission exists. In the very small particle limit the enhancement factor is given by [7] a a G i g r r i i g ag ag = + 3 i r r i r r r + r [ 3 0( 0. ) ] [ (. )] i (1.9) where refers to the polarization of the incident field at r, g and g 0 0 are the values of the function ( ε 1)/( ε + 2 ) evaluated at ω and respectively, ε is the ratio of the complex dielectric function of the material of the particles to that of the surrounding medium. If the adsorbed molecule is at a distance d from the surface of the metal particle, then r = ( a + d and according to equation (1.9) the enhancement is 0 ) 12 diminished as( a + d). ω / For a molecule situated on the surface of the sphere (i.e. r 0 = a) and with polarization of the incident and scattered wave perpendicular to the scattering plane the enhancement factor is given by [34, 35] 0 2 G=51+2g +2g+4gg 0 (1.10) When Re ε( ω) ~ -2 (which is the condition for the excitation of localized surface plasmons in the sphere), the quantities g and g 0 becomes large and in equation (1.10) the gg 0 term dominates and G becomes G = 80 gg0 2 (1.11) Thus the enhancement is large when both the frequency of the incident and the scattered beams approach the surface plasmon resonance condition and the quantity G becomes proportional to ε / // ε a r [36] where ε / = Re ε and ε // = Imε. Hence the metals having large ε / and small ε // values, at the frequency at which Re ε = -2, are expected to show large enhancement. That is why the alkali metals and the coinage metals are most suitable ones for SERS experiments producing G > In

22 13 addition, the plasmon resonance for these metals lies in or near the visible region of the spectrum. When the adsorbed molecules cover the entire surface of the sphere, the enhancement factor should be calculated by summing up the scattering from all molecules and averaging over molecular orientation. G is then given by [34, 35] 2 G = ( 1+ 2g)( 1+ 2g 0 ) (1.12) The mechanism involved in the enhancement remaining unchanged the enhancement differs when a molecule is adsorbed on a spheroid instead of being adsorbed on a sphere [29]. In such cases (1) The plasmon resonance shifts towards red. (2) The enhancement factor increases with the increase in aspect ratio (ratio of the semi major and the semi minor axes). (3) Metals that are not good plasmon enhancers may also show large enhancement if they are divided into spheroidal particles having large aspect ratio. (4) Hemispherical or hemispheroidal metal bump attached to a perfect conductor increases the enhancement 16 times [37] due to the imaging of the metal spheroidal dipole by underlying metal surface. For a molecule adsorbed on the hemispheroidal bump attached to a conducting plane, Gersten and Nitzan [38] calculated the enhancement factor as G 1 P P P = + ( 1 εξ ) 0 1( ξ1)/[ ε 1( ξ0) ξ0 1( ξ0)] 1 Γ 4 (1.13) Where a and b are semimajor and semiminor axes respectively of the spheroid and i) f = ( a b ) a ( a + d) ii) ξ0 =, ξ1 =, d is the distance of the molecule from the surface (on the f f axis of cylindrical symmetry) iii) ε is the complex dielectric constant of the metal iv) P 1 is the Legendre function of the second kind

23 14 v) Γ is a complex quantity and 2α Γ= [ 2( a+ d)] 3 in the limit ε 1, α being the molecular polarizability. The quantity Γ is not greater than 0.1 for d = 1 Å and thus the effect of term 1- Γ in equation (1.13) is less significant. If the ellipsoids become needle-like, then ξ 0 and ξ 1 approach unity, and G becomes larger. For larger aspect ratio G decreases rapidly with d i.e. for sharper surface features, the enhancement is larger only for the molecules directly adsorbed on the surface. In the Rayleigh small particle approximation, Wang and Kerker [39] calculated the Raman enhancement factor for prolate and oblate spheroids. They calculate the enhancement of the 1010 cm -1 band of pyridine with prolate silver spheroids of different aspect ratios. As the aspect ratio increases, the excitation wavelength corresponding to maximum enhancement shifts towards longer wavelength. This is partly due to the localization of the enhanced surface field around the tips of the spheroid. (ii) Collective Resonances In most real systems, the particles are very close so that one should consider the coupling of single particle resonances. The interaction between resonances is long range in character because the fields excited at resonance reach far (several hundred angstrom) outside the particle. For weak coupling regime the interparticle distances being larger, one expects frequency shifts and for the strong coupling case, completely new resonances occur. In between two successive particles, enormous field is created because the strong local field of one particle polarizes the other [40-42] and a molecule situated at this place is thus envisaged to show large enhancement of spectroscopic signals. Arvind et al [40] found that for a silver grating of period d = 1000 Å and λ = 5145 Å a resonance of the electric field strength occurs within the groove, intensity of which increases with decrease in groove width and the resonance frequency shifts to the blue with decreasing pore height. Arvind and Metiu [43] illustrated the physical effects of interaction between spheres with a sphere-plane system. For large distance between sphere and the plane, the only resonance one can excite by light is n = 1 dipolar resonance of the sphere (as discussed in the earlier section). Because of the momentum conservation, light cannot excite the surface

24 15 plasmon resonance of the flat surface but the dipole induced in the sphere can excite this resonance effectively. So in the presence of a sphere a mixture of n = 1 resonance of the sphere and a localized wave packet made up of high parallel momentum, plasmon states are excited when light is incident. Interaction between the sphere and its own image takes place when the sphere is brought close to the surface and this leads to n > 1 resonance of the sphere. This resonance interacts with the flat surface plasmon and thereby exciting it more. When the distance between the sphere and the plane is very small, these effects create a strong local field between the sphere and the plane surface. For two spheres very close to each other, similar interaction takes place. Liver at al [44] extended the calculation of Arvind et al [40] for more realistic SERS systems. They consider the field strength at different points in between and outside one-, two- and four-sphere assemblies. Their finding is that at a point in between two spheres the field is greater than that for a single sphere or at a point outside the sphere assemblies by more than an order of magnitude. This implies that for a molecule located at interstitial points the SERS enhancement is greater by four orders of magnitude. Moreover, for aggregated colloids they observe a red shift of frequency at maximum enhancement. Extrapolation of these results to aggregated colloidal assemblies having interstitial locations abound, indicates that the surface Raman signal is dominated by the molecules adsorbed at these sites. That for the twosphere assembly the field strength is greater than that for the isolated sphere explains the large SERS signal from slightly aggregated colloids. Inoue and Ohtaka [45] arrived at similar conclusions by considering only dipolar coupling between metal particles. This two-sphere cluster model has also been used to account for the depolarization, commonly found in SERS [46], for completely symmetric Raman active vibrations. The relationship, that exists, between the absorption spectrum and the SERS excitation profile for metal colloids or island films may be derived from the boundary condition relationship between the fields inside and outside the metal particles [47]. This is in agreement with the experimental observations [3, 11] with gold and copper colloids. For molecules adsorbed on roughened surface [48] and on metal colloids [11, 49], it is found that the Raman bands representing completely symmetric vibrations are notably less polarized compared to that for the free molecules. The depolarization ratio, for a particular mode of vibration, is a function of the relative magnitude of the

25 16 field components at the surface and the derived polarizability tensor component. The theoretical results based on the above-mentioned electromagnetic models are in poor agreement with the experimentally observed depolarization ratios for aggregated colloids and for other SERS active surfaces, (the Mie scattering from the aggregated colloids is also depolarized). Creighton [50] suggested that the depolarization of the Raman scattered light from molecules adsorbed on coagulated metal sols, is due to local electromagnetic anisotropy of the individual spheres within the aggregates. This concept has been further supported by the findings of Jiang et al [46] (iii) Quadrupole polarizability mechanism When a SERS active surface is illuminated near the plasmon resonance frequency, the field is enhanced and there is an optical frequency field gradient. These surface field gradients, in the case of a small sphere, exist surrounding a hypothetical point dipole at the centre of the sphere and are large for illumination at plasmon resonance frequency. The field is strongly localized between two closed spheres where the surface field gradient becomes larger and contributes significantly to SERS intensity. Sass et al [51] suggested that these optical frequency field gradients have significant contribution to the induced molecular dipoles at the surface via the molecular quadrupole polarizability, A given by 1 µ = αe + AE 3 (1.14) Where µ is the induced dipole moment,α is the molecular dipole polarizability, E is the incident electric field and E is the surface field gradients. In the SERS of benzene [52, 53] and ethylene [54, 55], some bands appear those are not usually Raman active. Reduction of symmetry of the molecule in the surface adsorbed state can apparently rationalize the appearance of such bands. However, Moskovits and DiLella [53] questioned about this concept. Most SERS spectra contain Raman forbidden bands [53, 56, 57] with slight frequency shift but large SERS intensity. If the concept of symmetry reduction holds, then slight frequency shift implies small perturbation but the large intensity of the forbidden modes suggests a very large perturbation of the molecule by the surface. In terms of the influence of the surface field gradients in inducing dipole moment through quadrupole polarizability of the molecule, the appearance of the Raman forbidden

26 17 bands can be explained more convincingly. It is further supported by the observation that dipole forbidden but quadrupole allowed electronic transitions are present in the fluorescence spectra of some polyenes at SERS active silver surfaces [58] (iv) Molecule in a trap: Multipolar treatment When the edge-to-edge separation between two spheres of the same size is smaller than their radius, the dipolar approximation fails and the excitation of higher orders multipoles must be taken into account [59]. Complete multipolar theory for the Raman response of molecules when place in between two spheres was proposed for the first time by Rojas et al [60]. The red shift of the plasmon resonance by the multipolar terms is enough to place it in the optical region. centered at z Consider a molecule (indexi = 2) at z = 0 placed in between two spheres = D and z = D (labeled i = 1and i = 3 respectively). For simplicity, spheres are assumed identical. An external electric field E 0 parallel to the excites the systems at frequencyω. In terms of multipolar moments excited in both spheres, the local electric field at the site of the molecule is given by z axis E L 4π 2 q ( ω ) = E0 + ( l + 1) l l + = ( 1) D q l+ 1 l, 1 l, 3 l+ 2 (1.15) Where q li,, the multipolar moments, appropriate for a three particles system. The local field is written as EL ( ω) = g 1 ( ω) E 0 (1.16) Where g 1 ( ω ) is now given by g 1 ( ω) = 1+ l= 1 3 i = 1 l ( l+ 1) a α D l+ 2 a i 1, i 1 1, i l ,i [( T ) l, 1 + ( 1) ( T ) l, 3 ] (1.17) Here T 1 is the inverse of the matrix T defined as l, i T li, ~ = δ δ B (1.18) l, i ll, ii, li, α 1,i is the dipole polarizability of the particle i [ α E 12, = α m ( ω) ], and a is its radius, i and we have assumed a a, a = R. Local field is considered at the molecular 1 = 3 2 0

27 18 sites and the equation (1.17) includes the effect of the latter only through the intermediacy of the spheres and this is accounted for by terms with i = 2. I The inelastic dipolar polarizability α ( ω, ω is independent of the m R ) magnitude of local field. Then its dipole moment is given by ( ω ) α ( ω ω) g ( ω) E 0 (1.19) I P m R = m R, 1 where g 1 ( ω ) is now the three particles fully multipolar field factor given in equation (1.17) The total dipole moments P ( ω, ω v ) responsible for the Raman signal is the sum of the dipole moments induced in the three particles at frequency ω R and is proportional to the dipole moment P m ( ω R ) of the molecule. By using equation (1.19) the total dipole moment may be written as I P( ωω, ) = g 1 ( ω) g 2 ( ω ) α ( ω, ω) E 0 (1.20) v R m with g 1 ( ω ) given by the equation I.16 and g 2 ( ω R ) is R g ai 1 ( ω R ) = + ( S ) a 1 1, i l= 1 i= 1 i = 1 2 li, ~ B 1, 2 li, (1.21) where the matrix S 1 is the inverse of the matrix S defined as where ~ li ~ l, i S li = δ, ll, δ ii, Dli, L ~ l, i ~ l, i ~ l, i~ D = B + B B li, l, i li, li, K, 2 K= 2 (1.21a) (1.21b) and the matrix B is defined as ~ B 1 l l, i 2l ai li, = l 2l + 1 i a B l, i li, (1.21c) The overall enhancement factor of the Raman intensity due to the presence of spheres is given by G g ( ω) g ( ω R ) (1.22) = 1 2 2

28 19 with g 1 ( ω ) and g ( 2 ω ) R given by equation (1.17) and (1.21), which include multipoles of all orders. (v) SERS on Flat surfaces: Image field theory Several groups [31, 61, 62] were reported the enhancement mechanism that would result from the electromagnetic interactions between a molecule and flat surface. In a popular model proposed by King et al [61], the electric field E of the radiation induces an oscillating dipole moment in the adsorbate molecule, which in turn, induces an image dipole in the metal. This image dipole has an associated electric field E im, which along with the local field enhances the overall field experience by the molecule. µ = α( E + E im ) whereα is the polarizability, and E im is given by: E im = ( ε ε 0 ) µ 3 ( ε + ε 0 ) 4r (1.23) where r is the distance between the dipole and the surface. Substituting and rearranging, one obtains, α ε ε µ = α 0 1 4r 3 ε + ε0 1 This equation has pole at the frequency at which E (1.24) αε ( ε) 0 1 Re = 1 3 ε + ε0 4r (1.25) Clearly, this is related to surface plasmon excitation in the metal surface, at the frequency for which the condition is Re( ε ) = ε 0. Thus, near the surface plasmon resonances, effective polarizability is increased and thereby resulting in large enhancement factors. Thus, in this model the enhancement is attributed to the increase of the apparent polarizability by the near zone image field of the induced dipole moment. This particular Coulomb interaction of a molecule, considered as a point dipole, with metal surface is extremely sensitive to the metal-dipole separation leading to large enhancement only for species very close to the surface. However, it requires no surface roughness or particular bonding. King et al [61] have shown that

29 20 on silver surface, Raman enhancement in excess of 10 6 are possible at or below a metal-dipole separation of 1.65 Å, and enhancement reduces to 10 2 for r=2 Å. (vi) Recent Development on the electromagnetic mechanism of SERS Recently Gracia-Vidal et al [63] reported a collective theory on SERS. They, for the first time, made it possible to handle surfaces consisting of complex particles close enough to interact strongly. A fully retarded implementation allows treatment of both large as well as small particles. They modeled a rough silver surface as an array of half cylinders embedded in a silver surface. They showed that the very much localized plasmon modes, created by strong electromagnetic coupling between touching metallic objects, dominate the surface enhanced Raman scattering response. Single molecule SERS in colloidal aggregates [64] was explained by Shaleav and co-workers [65] using different model. In this model, the particles in fractals are treated as dipoles with a resonance at the dipolar plasmon resonance polarized by the local fields consisting of the incoming laser field and the dipolar fields of the other particles. The model provides good qualitative insight into the random distribution of hot spots, quickly changing position with wavelength, direction and polarization of the incident light [66]. Very recently, Meyer et al [67] explained theoretically the phenomenology and statistics of single molecule SERS signals under the presence of electromagnetic Hot-spot. As the un-retarded fields are employed, no radiation damping is included and the calculated enhancements of the local fields may be very high. Any valid model of hot spots must account for the observation that only those molecules contribute to the observed SERS intensity, which alter the dc resistance of the cold-deposited films. Only direct adsorbed molecules do change the dc resistance [68, 69]. Hence, only contact adsorbed molecules contribute to the SERS spectrum. According to the report of Shalaev et al [65], the local electric fields in a semi continuous metal film exhibit giant fluctuations in the visible and IR spectral ranges, when the dissipation in metallic grains is small. The field fluctuations result in significantly enhanced Raman scattering from semi continuous metal films. They performed the scaling analysis to describe giant Raman scattering near the percolation threshold. Recently Etchegoin et al [70] is revisited the electromagnetic contribution in SERS. They discussed two different issues related to surface plasmon resonance: i)

30 21 the formation of the hot spots and the meaning of resonance, and ii) the existence of different scattering cross section for Stokes and anti-stokes SERS. However, Micic et al [71] reported the simulations of the influence of different surface morphologies on electromagnetic field enhancements at rough surfaces of the noble metals. Although Liu et al was found that electromagnetic enhancement plays an important role in the SERS of the palladium [72]. Also Gracia-Ramos et al [73] calculated direct electromagnetic enhancement in SERS on random self-affined fractal metal surfaces. They estimated the scattered electromagnetic intensity by means of numerical calculations based on the rigorous integral equations free from the limitations of electrostatic and/or the dipolar approximation. They reported that the fractal structure favors the occurrence of large electromagnetic enhancement, maximum at an optimum wavelength due the compromise between roughness-induced light coupling into surface plasmon and absorptive losses. (vii) Inadequacy of the electromagnetic model in explaining the overall SERS phenomenon The classical electromagnetic theory is undoubtedly mature enough to explain the enormous enhancement factor in SERS. However, several investigation as stated below, indicate that some other mechanisms also contribute significantly to SERS of which the chemical effect that involves electronic interaction between the molecule and the metal surface is the most important one. The observations that need the presence of such an interaction are as follows. (i) In a number of SERS experiment with colloidal metal particles it is reported that the excitation profile does not match with the absorption spectra of the sol [74, 75]. Creighton et al [74], however, found that the addition of pyridine solution to silver colloids gives rise to a shoulder in the absorption spectra of the colloids in the nm wavelength regions. They attributed it to the aggregation of the Ag particles in presence of pyridine. The excitation profile coincides with these longer wavelength features. (ii) Attempts have been made to explain the appearance of certain Raman forbidden bands, in SERS spectra, in terms of quadrupole polarizability mechanism [section a (iii)]. Nevertheless, according to Hallmark and Campion [76, 77], the quadrupole polarizability mechanism is neither necessary nor sufficient to explain their experimental observations. They emphasized on the symmetry reduction of the

31 22 molecules in the surface adsorbed state. Moskovits et al [78], with the help of their experimental results, concluded that the field gradient could explain the appearance of forbidden bands better than the symmetry reduction model. However, they did not rule out the possibility of symmetry reduction of the strongly bonded molecules. It is well agreed that the orientation of a molecule on the metal surface is an important factor to determine suitable surface selection rules [79]. Orientation of the molecules is different for different molecules and involves electronic interaction between the molecule and the metal surface. Allen and Van Duyne [80] explained the SERS spectra of cyanopyridines in terms of different orientation of the isomers depending on possible electronic charge transfer between the molecule and the surface. Bunding et al [81] found similar observations with isomeric methyl pyridines. Lombardi et al [82] confirmed the charge transfer interaction for substituted pyridines and for saturated nitrogen heterocyclic compounds. They found the charge transfer depends on the electronic charge density on the N atom of the molecules. (iii) The first layer effect which means maximum enhancement of Raman bands when a monomolecular layer is formed on the surface, cannot be successfully explained in terms of the electromagnetic mechanism only. Because this mechanism implies that, the enhancement effect is long range and the theory of this mechanism is based on the bulk dielectric constants. However, the short-range first layer effect can only be explained in terms of the electromagnetic mechanism if the roughness features are assumed to be smaller than 50 Å in size [83]. To quote Otto [84] there exists a short range enhancement mechanism confined approximately to adsorbates within the first directly adsorbed monolayer in addition to the long range classical electromagnetic enhancement. (iv) According to the electromagnetic model both CO and N 2 adsorbed on same metal surface should show almost same enhancement factor. However, the experimental results [85] indicate that CO shows larger frequency shifts and higher enhancement factor, which is an indication of chemisorption. (v) Plasmon resonances that enhance both the incident and the scattered light are strongly dumped in low reflectivity metals like platinum [86]. However, benzene is observed to undergo SERS when adsorbed on small platinum particles. [87]

32 23 (vi) For molecules like pyridine and pyrazine adsorbed on silver [88, 89], low energy charge transfer bands are observed in high-resolution energy loss spectroscopic experiments. Furtak and Macomber [90] also demonstrated the dependence of the SERS intensity of pyridine, adsorbed on silver electrode, on electrode potential for different excitation wavelengths, which is a very concrete evidence of charge transfer between pyridine and metal surface. (vii) Correlation of Raman enhancement of the molecules with their Hammett σ parameters suggest that charge transfer (CT) between the metal and the molecule makes a significant contribution to surface enhancement [91]. b) Charge-transfer mechanism of SERS The observations, which cannot be explained by the Electromagnetic mechanism alone, strongly suggest the existence of an additional mechanism in the SERS signal enhancement. Gersten et al [92] were the first group of researchers who proposed in 1979 this additional mechanism, generally termed as the charge-transfer (CT) mechanism or chemical mechanism. Mainly two types of CT interactions have been proposed: (i) Ground state charge transfer, (ii) Excited state charge transfer. (i) Ground state charge transfer The ground state charge transfer (CT) may also give rise to enhancement of Raman signals as shown by Aussenegg and Lippitsch [93]. According to them charges may be transferred between the molecule and the metal surface depending upon the distance between them. The molecular vibration modulates this CT and corresponding change in the polarizability depends strongly on the vibrational coordinate resulting in large enhancement of the associated molecular vibrational mode in the SERS spectra. Other researchers propose similar mechanism also [94-96]. A somewhat different type of ground state CT mechanism has also been reported by Otto [9]. Their concept is that the in-phase coupled vibrations of groups of surface molecules push and pull electron density to and from the surface, which modulates the surface reflectivity. However, the ground state CT model leads to the formation of surface-molecule complex. According to this model, the enhancements are expected only for the vibrations that are symmetric with respect to the symmetry elements of this complex. (ii) Excited state charge transfer

33 24 The other CT model suggests that an electron from a state below the Fermi level in the metal is excited to an unoccupied state of the adsorbed molecule [97, 98]. One can expect resonance if the energy of the incident photon matches with the CT transition energy. Avouris and Demuth [89], Schmeisser et al [99] confirmed the existence of such charge transfer excitation by electron-energy-loss-spectroscopy (EELS). They observed a broad band starting at 1.4 ev and peaked at 2.3 ev for pyridine adsorbed on silver. For a metal (D) to molecule (A) charge transfer, the CT contribution to the polarizability derivative of the system with respect to a normal coordinate Q A of the molecule as estimated by Avouris and Demuth [89] is given by δα δq toatal A δq A δα A δα A δα D = δr δqa δqa δqd CT (1.26) where α is the Raman polarizability of a metal-molecule system, q represents charge and R the donor-acceptor separation. The suffixes A and D denote the acceptor (usually the adsorbate) and the donor (usually the metal). The charge transfer (CT) mechanism of SERS can be explained by the resonant Raman mechanism in which charge transfer excitations from the metal to the adsorbed molecule or vice versa occur at the energy of the incident laser frequency [4, 100]. Campion and Kambhampati [4] proposed a hypothesis to understand the CT mechanism of SERS. The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of the probe molecule are symmetrically disposed in energy with respect to the Fermi level of the metal (Figure 3). Figure 3: Typical energy level diagram for a molecule adsorbed on a metal surface. The occupied and unoccupied molecular orbitals are broadened into resonances by their interaction with the metal states. Orbital occupancy is determined by the Fermi energy. Possible charge transfer excitations are shown.

34 25 In this case, charge transfer excitations (either from the metal to molecule or vice versa) can occur at about half the energy of the intrinsic intramolecular excitations of the adsorbate. In their reports they explained the observations by a resonance Raman mechanism in which either (a) the electronic states of the adsorbate are shifted and broadened by their interaction with the rough surface or (b) new electronic states which arise from chemisorption serve as resonant intermediate states in Raman scattering. Both Franck-Condon (FC) factor and Herzberg-Teller (HT) couplings are operative in CT mechanism [86]. Overlap of different vibrational substrates of the ground and excited electronic states are considered in the FC mechanism and mixing of different vibrational states by vibrationally induced changes in the electronic wave functions results in the HT coupling. The calculated enhancement factors are very small compared to the experimentally observed values, which indicates that the CT mechanism usually operates in conjunction with the electromagnetic effects. Persson considered Newns-Anderson resonances of an adsorbate on a silver urface and calculated the ratio of Raman scattering intensity of the adsorbed molecule with respect to the free molecule. [98] The discrete energy level of the lowest unoccupied orbital of the adsorbate is shifted and broadened into a resonance by short time excursions of electrons from the metal into this orbital a is equivalent to a partial filling of this broadened level by electrons. The charge transfer interaction involves the excitation of an electron from a metal state below the Fermi energy to about the maximum of ρ a where ρ a denotes the electron density of the adsorbate. The phonon-electron interaction is simply given by, H = ede n (1.27) photon electron z Q where d is the distance between the centre of charge of the orbital a and the E z metal image plane; is the incident field normal to the surface and n Q is an operator which describes the number of electrons in the orbital a. The molecule-vibration interaction is given by, H electron vibr = ε a ( ) Qn Q 0 (1.28)

35 26 where ε a is obtained from the expansion of the energy ε a of the orbital a of the free molecule with respect to the normal co-ordinate Q of the vibration. ε ( Q) = ~ ε + ε () 0 Q+ (1.29) a a a The chemical contribution to the enhancement factor, G chem, is obtained by Persson as 2 G chem 2 G( ω L, ω) = ( ed) ε a ( 0) 1 α ( 0) 2 (1.30) α ( 0) to Q is the derivative of the electronic polarizability of the free molecule with respect and this term governs the ordinary non-resonant Raman scattering, ω L is the incident laser frequency, ω is the Stokes frequency and G( ω, ω ) contains the resonant Raman scattering caused by the charge transfer interactions L (iii) Recent Reports on the Charge Transfer Mechanism Recently a new mechanism for the chemical contribution to the surface enhanced Raman scattering explained by Persson et al [101] The theory considers the modulation of the polarizability of a metal nanocluster or a flat metal surface by the vibrational motion of an adsorbed molecule. The modulated polarization of the substrate coupled with the incident light will contribute to the Raman scattering enhancement. Single molecule and single-particle studies have yielded great important insights into the mechanism of electromagnetic enhancement rather than the contributions of surface active sites and chemical enhancement. Nie et al [102] examined the chemical enhancement by using an integrated flow injection and ultra sensitive optical spectroscopy system. A key feature is that colloidal nanoparticles are immobilized on a glass surface inside a micro flow device and the single particle SERS signals are observed. They estimated the chemical enhancement factors are to be for rhodamine 6G molecules adsorbed on single Ag nanoparticles. A procedure to interpret SERS spectra has been developed by Otero et al [103] in order to clarify the controversy concerning the relevant enhancement mechanism of this type of spectra. The presence of charge transfer (CT) enhancement mechanism is detected by correlating the most enhanced SERS bands with the ab

36 27 initio calculated geometries and vibrational frequencies of the isolated molecule and its radical anion. This CT mechanism is supposed to be analogous to that in Resonance Raman (RR), an electron being transferred from the Fermi level of the metal to vacant orbitals of the adsorbate. Recently Otero et al proposed a model concerning the role of the electrode potential on the charge-transfer enhancement mechanism of SERS [104]. Campion and his co-workers have investigated the mechanism of the chemical enhancement of SERS [ ]. They reported that a number of molecules bound to the surfaces of Cu (111) and Cu (100) in ultra high vacuum produce Raman scattering that is 10~100 times more intense than that predicted on the basis of their intrinsic Raman scattering cross sections and surface coverage. The frontier orbital theory plays a significant role in the understanding of the molecule to metal or metal to molecule CT mechanism of SERS. Molecule to metal CT excitations occurs when an electron is transferred from the highest occupied molecular orbital (HOMO) of the adsorbate to the Fermi level (E F ) of the metal. Conversely, transfer of an electron from the E F of the metal to the lowest unoccupied molecular orbital (LUMO) results in metal to molecule charge transfer. [ ] SERS selection rule The selection rule, which should operate in SERS, if the source of enhancement is primarily electromagnetic in origin and to the extent that SERS active surface may be approximated to spherical colloidal metal particles, small with respect to the wavelength of light, have been worked out by Moskovits [ ]. The potential about which the sphere having a dielectric function ε immersed in an ambient of dielectric constant ε 0 and exposed to a radiation whose incident field strength is E 0 polarized in the Z direction is given by where 3 gr Vr (, θ ) = E0 r cosθ r g = ( ε ε )( ε + ε ) 2 (1.31) (1.32)

37 and R is the radius of the sphere. The square moduli of the tangential ( and the normal field ( components averaged over all solid angles at the surface of the E n sphere ( r = R) are given by E 2 t ) 2E 1 g (1.33) E = E 1+ 2g (1.34) n The electromagnetic SERS intensities are calculated as the product of the field intensities associated with the incident and the Raman scattered beams. If the molecule is adsorbed in such a way that the Z axis of the molecule-fixed co-ordinate frame lies along the metal surface normal, then three classes of vibrational modes are predicted to have the following SERS enhancements: ( E )( E ) 1+ 2g 1+ 2 α (1.35) zz : n n g [ En Et + E E t n ] ( 1+ 2g 1 g + 1 g 1+ 2 ) 1 α XZ, αyz : g (1.36) XX, α YY, α : E E XY t t 4 1 g 1 g α (1.37) E t ) 28 Here the prime indicates the properties to be calculated at Raman shifted frequency. On satisfying the surface plasmon resonance condition, Re( ε ) = 2ε 0, and g >> 1. Thus at and to the red of surface plasmon frequency 2 ( E n ) exceeds (E t 2 ). Under these conditions, SERS spectrum will be dominated by the α ZZ modes. The α XZ and α YZ modes will be less intense, and α XX, α and α XY modes will be the least intense SERS of Molecules adsorbed in Langmuir-Blodgett (LB) Film Cipriani [112] with his group was the first who reported the Raman spectroscopy in multilayer films. They studied LB multilayers of barium stearate in a total reflection mode. Lis et al studied Raman spectra of LB multilayers of behnic acid, barium behenate and barium cis-13-erucate [113]. Robolt et al [114] demonstrated for the first time the use of integrated optics in Raman scattering YY

38 29 studies. Here, the material used into an asymmetric slab waveguide, or a composite waveguide structure in which both the optical field intensity of the in-coupled laser source and the scattering volume of the sample were increased significantly. Using this technique, they obtained Raman spectrum of a single monolayer of a dye. Chamberlain et al [115] presented a new approach for the acquisition of Raman spectra of Langmuir monolayers. They generated colloidal silver electrochemically under the layers of anionic surfactants stearic acid and dipalmitoylphosphatidic acid sodium salt on an aqueous AgNO 3 subphase. Knoll et al [116] reported for the first time the monolayer SERS spectra of cadmium arachidate in contact with variety of rough silver surfaces. Aroca et al and de Daja et al [ ] put forward the SERS of substituted phthalocyanine molecules organized in LB films. They reported the evidence of electromagnetic mechanism in SERS, where LB films of arachidic acid were used as a spacer layer to control the separation distance between the evaporated indium and silver metal films and LB monolayers of the phtalocyanine molecule. Their results rule out the shortrange chemical mechanism, i.e., an interaction between the metal atoms and the phthalocyanine molecule. LB-SERS spectra of perylene derivative [ ], metal-free porphyrazine and its copper complex [123] and fullerenes (C 60 ) [124] are reported to elucidate the vibrational analysis and structural details of these molecules. From all these reports, it is concluded that the physisorption between the LB monolayers and the metal island film are the main mechanism of enhancement. Menendez et al [125] reported the SERRS intensity pattern versus surface molecular coverage, both at room temperature and at low temperature using LB monolayers of tetra-tert-butylvanadyl-phthalocyanine and luthetium bisphthalocyanine with arachidic acid. SERS imaging and mapping of Langmuir- Blodegtt monolayers of bis-(n-butylimido)perylene on silver island films are reported by Aroca et al [126]. They utilized the micro-raman imaging technique to achieve the visualization of SERRS signal from silver islands coated with LB monomolecular layer. Very recently Aroca et al reported SER spectra of LB films of an azopolymerpyridine.[127] Yang et al was used LB technique to assemble monolayers of aligned silver nanowires that are ~ 50 nm in diameter and 2-3 µm in length. The resulting

39 30 nanowire monolayers serve as excellent substrates for surface-enhanced Raman spectroscopy with large electromagnetic field enhancement [128] Fibre-Optical SERS As Raman spectroscopy leaves a vibrational signature of molecules using visible light, it is well suited for use with optical fibers, which transmit light with high frequencies. Mostly the fibers used for Raman spectroscopy typically consist of fused silica due to its small transmission losses in visible light and very little fluorescence. Optical fibers are used extensively in normal Raman spectroscopy [129], but the first fiber-optic SERS sensors were developed by Bello et al [130] Mullen et al reported SER spectrum by putting a SERS-active surface directly at the end of the excitation fiber by depositing thermally evaporated silver on an abrasively roughened fiber tip [131]. So far, only a few approaches have been made to achieve surface preparations of fiber-optic SERS probes. Stokes et al [132] dip-coated the fiber tips in an aqueous suspension of alumina and McDonald et al [133] applied silver island films to fiber tips which they tested using the dye CoPc. Viets et al [134] compared fiber-optic SERS sensors with differently prepared tips. Recently Viets et al [135] observed that different angle of the tips of fiber-optic SERS sensors results in an additional enhancement of SERS intensities by factors of 3-20 for different tip coatings. In order to be able to optimize the SERS enhancement at fiber tips in a better way, a method is developed to prepare regular structure on fiber tips. Recently, grating structure has been patterned by electron beam lithography on fused silica fiber tips that has previously been coated with the resist PMMA [136]. Such structures are then transferred into the fused silica material by reactive ion etching and finally silver coated to achieve SER activity. The modification of SERS substrates with cage or cone-like molecules, like calixarenes [137] or cyclodextrins [138] yield, surfaces capable of adsorbing low concentrated aromatics from the gas or liquid phase Tip Enhanced Raman Scattering (TERS) It is well known that the SERS effect can improve the Raman signal by several orders of magnitude. However, a rather severe limitation of SERS is that the enhancement varies strongly with the substrate morphology and therefore with the lateral position and hence critically depends on the substrate preparation, which eventually makes quantitative measurements almost impossible.

40 31 Wessel et al [139] introduced a method of increasing the Raman signal due to the same surface enhancement effect, without preparing any special substrate. The rough metal film is replaced by one metal particle that is scanned over the sample using scanning probe microscopy (SPM) technique. Excitation and the collection of the Raman spectra were recorded with a standard Raman microscope set up. Any signal enhancement is expected to originate from the same grain, which provides a kind of uniform SERS substrate. The single metal particle was formed by evaporating a noble metal onto an atomic force microscopy (AFM) tip. Alternatively, a sharp solid metal tip was made directly by electrochemical etching of a thin wire. Either tip was mounted into a commercial scanning near-field optical microscope that was used as a scanning platform. The enhanced Raman scattering at the tip of the metal surface is popularly termed as tip enhanced Raman scattering (TERS or TSERS) Very recently, Zhang et al [140] reported the single molecule tip-enhanced resonance Raman spectra from brilliant cresyl blue (BCB) submonolayers adsorbed on a planar Au surface with Ag tips. A gap of 1 nm between a Ag tip and the Au substrate was employed to create a highly enhanced electric field and to generate Raman scattering from an area of ~ 100 nm 2. Also very recently, Domke et al [141] presents the TERS spectra of four DNA bases adenine, guanine, thymine and cytosine, respectively adsorbed homogeneously at Au (111) in picomole quantities, proves the applicability of TERS for biochemically hughly relevant, optically nonresonant species and highlights the sensitivity of this technique. This particular tip-enhanced Raman scattering (TERS/TSERS) technique may be useful to study the origin of SERS phenomenon in near future due to the fact that the laser wavelength can be tuned to exactly match the plasmon frequency of the single particle and due to combination of SPM techniques, distance dependence investigations can be easily done with high precision Single Molecule Detection Using SERS Detection of single molecule in solution with sensitivity and molecular specificity is of great scientific and practical interest in many fields such as chemistry, molecular biology, medicine and environmental science. SERS is a useful technique resulting in strongly increased Raman signals from molecules attached to nanometer sized metallic structures. Almost at the same time, Nie et al [142] and Kneipp et al

41 32 [143] published the first reports on single molecule detection using SERS. In the reports of Nie et al, the probe is a single Rhodamine 6G molecule, adsorbed on the selected silver nanoparticles using a laser line in resonance with the electronic absorption of the molecule. Therefore, the observed inelastic scattering corresponds to SERRS. However, in the reports of Kneipp et al, the enhanced Raman scattering spectra of a single Crystal Violet molecule in colloidal silver solution was obtained using nonresonant near-ir excitation, i.e. using a laser line with a frequency outside the electronic transition of the molecule. In both cases, observed enhancement in the range ~ to 10 15, which is much larger than the ensemble-averaged values derived from conventional measurements. This enormous enhancement leads to vibrational Raman signals that are more intense and stable than single-molecule fluorescence. Single-molecule SERS/SERRS brought about a renaissance of SERS activity with a great attention on the fabrication and properties of nanostructures that can provide the huge enhancement factor needed for single molecule detection (SMD). Xu et al [144] reported the detection of single hemoglobin protein molecule attached to isolated and immobilized Ag nanoparticles using SERS. SERRS from single myoglobin molecules was reported [145] and the vibrational spectrum of single horseradish peroxidase molecule was detected by measuring SERS from isolated and immobilized proteinnanoparticle aggregates. [146]. SERS of single-stranded DNA [147], SERRS spectra of Fe-protoporphyrin IX, adsorbed on Ag colloidal nanoparticles immobilized on a polymer-coated glass slide [108], and single molecule SERRS of the green fluorescent protein [148] have been reported. SMD is achieved by spatially resolved SERRS microscopy of a single Langmuir-Blodgett (LB) monomolecular layer containing dye molecules dispersed in fatty acid, i.e. a two dimensional host matrix fabricated using the LB technique [149]. At present near infrared, SERS has been utilized to detect single molecule and to specify hot vibrational transitions [150]. Very recently, Ru et al [151] proposed a new method to proof for single-molecule sensitivity in surface enhanced Raman spectroscopy. The simultaneous use of two analyte molecules enables a clear confirmation of single (or few)-molecule nature of the signals.

42 Quantum Chemical Calculations: Ab Initio and Density Functional Theory Methods a) Ab initio Method The presence of several nuclei in polyatomic molecules makes quantummechanical calculations harder than for diatomic molecules. Moreover, the electronic wave function of a diatomic molecule is a function of only one parameter, the internuclear distance. In contrast, the electronic wave function of a polyatomic molecule depends on several parameters- the bond distances, bond angles, and the dihedral angles of rotation about single bonds (these angles define the molecular conformation). A full theoretical treatment of a polyatomic molecule involves calculation of the electronic wave function for a range of each of these parameters. The equilibrium bond distances and angles are then found as those values that minimize the electronic energy including nuclear repulsion. The four main approaches to calculating molecular properties are ab initio methods, semi empirical methods, the density functional method and the molecular-mechanics method. However, in this thesis we use ab initio and DFT methods to calculate the different molecular properties. Ab initio calculations [152, 153] are used to calculate the minimum energy geometrical arrangement of the atoms in a molecule and vibrational frequencies of different molecular complexes. Use of the calculated quantities with simple statistical mechanical techniques allows the calculation of thermo-chemical properties, such as the enthalpy, entropy, free energy and equilibrium constants for various reactions. It has been demonstrated that in most cases, accuracy equal to that from experimental measurements is achievable. The term ab initio means from first principles it however, does not mean that we are solving the schrodinger equation exactly. It essentially means that we are selecting a method that, in principle, can lead to a reasonable approximation to the solution of the schrodinger equation and then selecting a basis set that will implement that method in a reasonable way. The essential idea of the method is that, for a closed shell system, two electrons are assigned at a time to a set of molecular orbitals. In order to have a freedom to vary the molecular orbitals that best suit the molecule in question, one can expand each

43 34 molecular orbital in terms of a set of basis functions, which are normally centered on the atoms in the molecule. The Hartree-Fock (HF) method has been widely used in the chemical sciences to form the basis of ab initio molecular orbital theory; this is an approximate method for the determination of the ground-state wave function and ground-state energy of a quantum many-body system. The HF method assumes that the exact, N-body wave function of the system can be approximated by a single Slater determinant (in the case where the particles are fermions) or by a single permanent (in the case of bosons) of N spin-orbitals. Invoking the variational principle one can derive a set of N coupled equations for the N spin-orbitals. Solution of these equations yields the HF wave function and energy of the system, which approximate the exact ones. The HF method is also called, especially in the older literature, the selfconsistent field method (SCF). The solutions to the resulting non-linear equations behave as if each particle is subjected to the mean field created by all other particles (see the Fock operator below). The equations are almost universally solved by means of an iterative, fixed-point type algorithm (see the following section for more details). This solution scheme is not the only one possible and is not an essential feature of the HF method. For molecules, HF is the central method for all ab initio quantum chemical methods. In the HF method, a single electron moves independently in the field of fixed nuclei and in the average Coulomb and exchange fields of all other electrons. Therefore, it is also known as the independent particle approximation. HF wave function is written as an antisymmetrized product of spin-orbitals, each spin-orbital being a product of a spatial orbital Ψ i and spin function α and β. The molecular orbital wave function for the closed-shell ground state of a molecule with n number of electrons (when n is an even number) is given as: ψ1(1) α(1).. ψ1(1) β(1) ψ2(1) α(1) ψ2(1) β(1) ψn (1) β(1) 2 ψ1(2) α(2) ψ1(2) β(2) ψ2(2) α(2) ψ2(2) β(2) ψn (2) β(2) 2 1 Ψ= ( n!) 2 ψ1(3) α(3) ψ1(3) β(3) ψ2(3) α(3) ψ2(3) β(3) ψn (3) β(3) 2 ψ1( n) α( n) ψ1( n) α( n) ψ1( n) β( n) ψ1( n) β( n) ψn ( n) β( n) 2

44 35 This determinant is popularly known as a Slater determinant. The individual molecular orbital in the above determinant is expressed in terms of a linear combination of finite sets of one-electron functions known as basis functions. Therefore ψ i = N µ = 1 C µ iϕ µ (1.39) Where, C µi are the molecular orbital expansion coefficients. The HF energy of closed shell molecular system with n electrons is written as: HF n / 2 n / 2 n / 2 core = 2 H ii + i = 1 i = 1 j = 1 E (2 J K ) + ij ij V NM (1.40) Where the first term on the right hand side is the one electron core Hamiltonian, the second one is the Coulomb integral term, the third one is the exchange integral term, and the last one is the nuclear repulsion term. The main deficiency of the HF method is the lack of treatment of correlation of electronic motions in the system. Particularly, the single determinant wave functions do not take into account the correlation between electrons with opposite spin. However, correlations between the motions of electrons with the same spin are partially accounted in terms of the determinantal form of the wave function. b) Density Functional Theory Method Density functional theory (DFT) is a quantum mechanical method used in physics and chemistry to investigate the electronic structure of many-body systems, in particular molecules and the condensed phases. DFT is among the most popular and versatile methods available in condensed matter physics, computational physics, and computational chemistry [ ]. The predecessor to DFT was the Thomas-Fermi model, developed by Thomas and Fermi in They used a statistical model to approximate the distribution of electrons in an atom. The mathematical basis used was to postulate that electrons are distributed uniformly in phase space with two electrons in every h 3 of volume. For each element of coordinate space of volume d 3 r, one can fill out a sphere of momentum space up to the Fermi momentum (p f ). After Solving for p f and

45 36 substituting it in the classical kinetic energy formula, leads directly to a kinetic energy represented as a functional of the electron density. It is possible then to calculate the energy of an atom using this kinetic energy functional combined with the classical expressions for the nuclear-electron and electron-electron interactions, which can both also be represented in terms of the electron density. Although this was an important first step, the Thomas-Fermi equation's accuracy is limited because the resulting kinetic energy functional is only approximate, and the method does not attempt to represent the exchange energy of an atom as a conclusion of the Pauli principle. Dirac added Exchange energy functional in The Thomas-Fermi-Dirac theory remained rather inaccurate for most applications however. The largest source of error was in the representation of the kinetic energy, followed by the errors in the exchange energy, and due to the complete neglect of electron correlation. In 1962, Teller showed that Thomas-Fermi theory could not describe molecular bonding. It can however be overcome by improving the kinetic energy functional by adding the Weizsäcker (1935) correction. Although density functional theory has its conceptual roots in the Thomas-Fermi model, DFT was not put on a firm theoretical footing until the Hohenberg-Kohn theorems (HK). [157] Traditional methods in electronic structure theory, in particular Hartree-Fock theory and its descendants, are based on the complicated many-electron wavefunction. The main objective of density functional theory is to replace the many-body electronic wavefunction with the electronic density as the basic quantity. Whereas the manybody wavefunction is dependent on 3N variables, three spatial variables for each of the N electrons, the density is only a function of three variables and is a simpler quantity to deal with both conceptually and practically. The most common implementation of density functional theory is through the Kohn-Sham method [158]. Within the framework of Kohn-Sham DFT, the intractable many-body problem of interacting electrons in a static external potential is reduced to a tractable problem of non-interacting electrons moving in an effective potential. DFT has been very popular for calculations in solid state physics since the 1970s. However, it was not considered accurate enough for calculations in quantum chemistry until the 1990s, when the approximations used in the theory were greatly

46 37 refined to better model the exchange and correlation interactions. DFT is now a leading method for electronic structure calculations in both fields. Despite the improvements in DFT, there are still difficulties in using density functional theory to properly describe intermolecular interactions, especially van der Waals forces (dispersion), or in calculations of the band gap in semiconductors. The development of new DFT methods designed to overcome this problem, by alterations to the functional or by the inclusion of additive terms, is a current research topic. The density functional theory (DFT) method does not attempt to calculate the molecular wave function but calculates the molecular electron probability density ρ and calculates the molecular electronic energy from ρ. In DFT, the exact exchange (HF) for a single determinant is replaced by a more general expression, the exchangecorrelation functional, which can include terms accounting for both exchange energy and the electron correlation that is omitted from Hartree-Fock theory. The three hybrid functionals, which include a mixture of Hartree-Fock exchange with DFT exchange-correlation, are available via keyword: B3LYP (Becke Three Parameter Hybrid Functionals). These functionals have the form devised by Becke in 1993 as: A * E Slater X + * * * ( 1 A ) E + B E + E + C E (1.41) HF X Becke X VWN C non local C Where A, B, and C are the constants determined by Becke [159]. There are several variations of this hybrid functional. B3LYP uses the non-local correlation provided by the LYP (Lee, Yang and Parr) expression [160]. It is to be noted that since LYP includes both local and non-local terms, the correlation functional actually used has the form: C * E LYP C * VWN ( 1 C) E + (1.42) C In other words, VWN is used to provide the excess local correlation required since LYP contains a local term essentially equivalent to VWN. The BPW91 method, which combines Becke s exchange functional [161] with Perdew-Wang 91 correlation functional [162], was also used for different molecular properties calculations. DFT calculations may be performed using different basis sets and basis sets used for calculations for different types. In general, the number and type of basis functions contained in them classify basis sets. They assign a group of functions to each atom within a molecule to

47 38 approximate its orbitals. Standard basis sets for electronic structure calculations use linear combination of Gaussian functions of the type g ( α, r ) cx y z e 2 m n l α r = (1.43) where α is a constant determining the size of a function, to form the orbitals and r is of course composed of x, y, and z. A linear combination of Gaussians is used to form actual basis functions, known as contracted functions, which have the form d χ (1.44) µ = µ p g p p where d µp are fixed constants within a given basis set. A basis set can be made larger by increasing the number of basis functions per atom. Thus, basis set like the one 6-31G [163] used in the present study is generated by increasing the number of primitives devoted to the core and first valence electron functions. This is called splitvalence basis sets. Split-valence basis sets allow orbitals to change size but not the shape. Polarized basis sets remove this limitation by adding orbitals with angular momentum beyond what is required for the ground state to the description of each atom. Thus polarized basis functions 6-31G ** also written as 6-31G(d, p) [164] used in the present study is formed by adding d and p functions to the heavy atoms and the hydrogen Atoms respectively. Another type of basis set i.e. Hay and Wadt basis set [165] with double-zeta quality (Lanl2DZ) have been used for modeled surface complexes. Lanl2DZ is an effective core potential (ECP) that includes mass-velocity relativistic effects for core electrons. In quantum chemical calculations, both RHF and DFT, due to the non-inclusion of the anharmonicity, the calculated force constants are generally higher by about 10-15%. Further, RHF calculations neglect exchange and correlation effects, which are included in DFT calculations. Hence, a suitable scaling factor has to be used to relate the calculated force constants to the experimental force constants Normal Coordinate Analysis The dynamical quantities of interest in an isolated molecule executing small vibrations are summarized by the classical secular equations. Under harmonic approximation, the classical secular equation relates the intra-molecular force field and forms of the vibrational motions to the molecular geometry, atomic masses and

48 39 spectroscopic frequencies associated with the fundamental modes of vibration. The general method of framing and solving vibartional secular equation is known as normal coordinate analysis. (i) Internal coordinates. The description of the motions of a polyatomic molecule having N nuclei requires 3N Cartesian coordinates represented by a column matrix X. Of these, six for a non-linear molecule and five for a linear correspond to translational and rotational motions. The remaining 3N-6 or 3N-5 coordinates are called Internal coordinates represented by the column matrix R. The vibrational energy of a polyatomic molecule, which is a sum of the kinetic energy T and potential energy V, can be expressed in different ways. It is chemically more intuitive to solve the vibrational problem in terms of internal coordinates (R) corresponding to change in bond lengths, bond angles, etc., rather than the Cartesian coordinates. The relationship between the two coordinate sets R = BX (1.45) where B is the transformation matrix, is linear only in the harmonic approximation. (ii) Equation of motion Wilson et al [166] expressed the equations of motion as FG Eλ = 0 (1.46) s As where F and G are the force constant and the kinetic energy matrices and A s is the column matrix of maximum vibration amplitudes ams corresponding to the frequency. The frequencies λ s and the corresponding eigen vectors A s can be obtained by solving equation. ν s The a ms value may be calculated, assuming their normalization such as m ( a ) 2 = 1 (1.47) ms The normalized vibration amplitude x ms is then a x (1.48) ms ms = 2 2 ( m( a ) ) 1/ ms

49 40 (iii) Vibrational normal modes and potential energy distribution In the harmonic approximation, all the vibrational modes are orthogonal, which means that they do not exchange energy. A system of coordinates Q s called Normal coordinates may then be found such that the kinetic and potential energy may be written as quadratic equations without the inclusion of interaction terms. The internal coordinates can be related to the normal coordinates by an expression R m = s L ms Q s where L is the transformation matrix. R = LQ (1.49) Using normal coordinates, the vibrational frequencies may be expressed by the 2 relation λ f L L f or s = + L m ms mm m m ms m s mm m 2 Lms λs f mm + m m LmsL λs m s f mm = 1 (1.50) The terms 2 L ms λ s and L ms L m s λ s are called potential energy distribution. The first term correspond to the potential energy of the normal mode ν s of the principle force constant f mm and the second term represents the contribution of the interaction force constant f mm. In general, Lms λ s terms are larger and provide a reasonable measure of the contribution of the internal coordinate R to the normal coordinate Qs. m Two-Dimensional Correlation Spectroscopy The concept of two-dimensional correlation spectroscopy was developed by Noda and has wide popularity, particularly among vibrational spectroscopists [ ]. The 2D correlation spectroscopy comprises, basically, two types of correlation spectra, the synchronous [φ (ν 1,ν 2 )] and asynchronous [ψ (ν 1,ν 2 )] spectra. The basic concept used to build 2D correlation spectrum is the analysis of dynamic spectrum i.e., the spectral analysis in the frequency domain of the spectral characteristics that changes in the time domain, due to external perturbations. 2D-correlation spectroscopy simplifies the investigation of complex spectra, enhancing spectral resolution by spreading peaks along the second dimension [169].

50 41 A perturbation-induced variation of a spectral intensity y (ν, t) is observed during a fixed interval of some external variable t between T min and T max. While this external variable t in many cases is the conventional chronological time, it can also be any other reasonable measure of a physical quantity, such as temperature, pressure, concentration, voltage etc., depending on the type of experiment. The dynamic spectrum y (, t ) external perturbation is formally defined as ~ ν of a system induced by the application of an y y( ν, t) y( ν) fortmin < t< Tmax ( ν, t) = { 0 otherwise (1.51) where y (ν) is the reference spectrum of the system. While selection of a proper reference spectrum is not strictly specified, in most cases, it is customary to set y (ν) to be the stationary or averaged spectrum given by y max ( ) = y ( ν, t )dt T max 1 T min T T ν (1.52) min X In general, the 2D correlation spectrum can be expressed as ( ν ) = ~ y ( ν, t ). ~ y (, t ) X ( ν ν 2 ν (1.53) 1, ) ( ) 1, 2 1 ν 2 represents the intensity of 2D correlation spectrum which quantitatively estimates a comparative similarity or dissimilarity of spectral intensity variations [ ~ y ν,t ] measured at two different spectral variables, ν 1 and ν 2, during a fixed interval. In order to simplify the mathematical manipulation, we treat X ( ν, ν ) as a 1 2 complex number function ( ν1 ν 2 ) φ( ν1, ν 2 ) iψ ( ν1, ν 2 X, + ) = (1.54) Equation (I.54) comprises two orthogonal (i.e. real and imaginary) components, whose real part represent the synchronous and the imaginary part depicts the asynchronous 2D correlation intensities respectively. A synchronous spectrum characterizes the similarity between the sequential variations of spectral intensities and is generally symmetric with respect to the

51 42 diagonal line corresponding to co-ordinates ν 1 = ν 2. Any region of the spectrum, which changes intensity largely under a given perturbation, will show strong auto peaks in the synchronous spectrum, while those remaining near constant develop little or no auto peaks. An asynchronous cross peak develops only if the intensities of two spectral features change out of phase with each other. The intensities of asynchronous peaks signify the sequential or unsynchronized changes of spectral intensities measured at correlated frequencies. This feature is especially useful in differentiating overlapped bands arising from spectral signals of different origin. 2D correlation spectroscopy has been successfully applied to IR [170], NIR [171], Raman [ ], ultraviolet-visible (UV-vis) [174], fluorescence [175], circular dichroism (CD) [176], and vibrational circular dichroism (VCD) spectroscopy [177]. 2D correlation spectroscopy is not even restricted to optical spectroscopy but it has been also applied to X-ray [178] and mass spectrometry [179]. We have utilized the 2D correlation spectroscopic technique to envisage the preferential nature of the adsorbed species of molecules on the nano colloidal surface Applications of SERS in areas of contemporary interest The recent up thrust in the field of SERS is mainly in three directions: understanding of SERS mechanisms (whose exact origin is still not clear), the search for new nano structured SERS active substrates and use of SERS for imaging and the search for new applications. Apart from the numerous reports on the application of SERS in the elucidation of the structural details and surface chemistry of complex molecules, this phenomenon is now successfully utilized in recording the Raman spectra of various systems. There are many reports such as single-walled carbon nanotubes [180], C-70 adsorbed on roughened silver surface [181], detection of atmospheric contaminants in aerosols [182], vibrational signatures of graphite [183], SERS of water in hydrogen evolution reaction [184]. SERS technique was used for investigation of trace interfacial water at silver electrodes in a series of normal alcohols [185], effect of chiral β-blocker drugs on colloidal silver [186], DNA triple helix [187], and many other molecules at trace concentrations. In addition, SERS technique has been utilized in understanding the specific interactions of antiretroviraly active drug hypericin with DNA [188], bacterial lipolysis in a skin pore phantom

52 43 [189], the stability and control of protein orientation using protein: colloid conjugates [190], the detection of nicotinamide in vitamin tablets [191]. Cinta et al [192] reported the SERS mechanisms of Vitamin PP in aqueous silver and gold colloids. Nowadays different types of substrate are used for SERS. Fang et al [193] reported SERS spectra of C 60 /gold nano cluster and C 70 /gold nano clusters deposited on floppy disk and hard disk. In addition, Niu et al [194] reported the SERS spectra of single-walled carbon nanotubes (SWCNTs) on metal-coated filter paper. SERS phenomenon has now been successfully utilized in single molecule imaging [195] and in the detection of a single living cell [196]. With the aid of nano-science (technology) numerous methods have been developed to synthesize nanoparticles of controlled size and shape and to fabricate nanorod or nanodot arrays, and hence to obtain SERS active surfaces. Very recently, a spectroscopic assay based on SERS using silver nanorod array substrates has been developed that allows for rapid detection of trace levels of respiratory Viruses with a high degree of sensitivity and specificity [197]. Biocompatible, photostable and multiplexing-compatible surface enhanced Raman spectroscopic tagging materials composed of silver nanoparticles-embedded silica spheres and organic Raman labels for cellular cancer targeting in living cells have been reported [198]. The SERS effects obtained by gold droplets on top of Si nanowires have been reported recently [199] Limitations of the SERS technique Despite the enormous advantages of sensitivity and selectivity associated with SERS, a number of significant limitations of this approach do exist. The foremost among these is that the intense SERS signals can only be observed on a discrete set of metal surfaces while surface infrared spectroscopy can be applied to any kind of surface. A second problem with SERS approach is that the relative contributions of different enhancement processes are not characterized well. It is now widely accepted that the enhancement of electromagnetic field near the metal surface and the light induced excited state metal-surface charge transfer are the two main causes of SERS. However, the magnitude of charge-transfer enhancement associated with atomic scale roughness features can change with surface pretreatment, applied potential and excitation wavelength. Further, the surface selection rules derived from the two

53 44 models are usually contradictory and no general rule is available at present to predict the surface orientation of an adsorbate reliably from its SERS spectrum. This is in contrast with other surface sensitive techniques such as surface infrared and EELS, for which the selection rules are well established.

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65 56 CHAPTER 2 Experimental Section and Computational Software 2.1 Introductory Remarks SERS is a recent addition to various established surface sensitive spectroscopic techniques [1-5] such as Infrared transmission, absorption or reflection spectroscopy, electron tunneling spectroscopy etc., which of course, has developed into a diagnostic probe for the analytical characterization of the adsorbates and microscopic structure of surfaces and interfaces. In earlier chapter, a brief introduction on various aspects of SERS has been presented. In this chapter, a discussion on the methods of sample preparation, purification and various experimental techniques of NRS, SERS, FTIR etc. is presented. 2.2 Preparation of Silver Nanocolloid Silver nanocolloidal solution was prepared following the process described by Creighton et al [6]. Silver nitrate and sodium borohydride were purchased from E. Merk (Germany). 1.0 x 10-3 M solution of AgNO 3 and 2.0 x 10-3 M solution of NaBH 4 were prepared with deionized distilled water (using Milli Q plus system of Millipore, U.S.A.). 15 ml of NaBH 4 solution was ice cooled and stirred vigorously with the help of a magnetic stirrer. 5 ml of AgNO 3 solution was then added rapidly. The yellowish sol, thus prepared, was stored at 5 C. The yellow sol shows a single extinction maximum at 392 nm and was stable for several weeks. Care has been taken with the purity of the water used and cleanliness of glassware to make the prepared sol to stable. 2.3 Sample Preparation and Purification All samples were purchased from Aldrich Chemical Co. U.S.A., and were further purified by recrystallization method. The three Benzothiazole derivatives molecules (2-aminobenzothiazole, 2-amino-4-methylbenzothiazole, 2-amino-6- methylbenzothiazole) are insoluble in water but are readily soluble in acetonitrile

66 57 solution. The solvent used is of spectroscopic grade and is free from impurities. The Raman bands of these samples were normalized with respect to the acetonitrile band. Rhodamine 123 and 4-methyl-4H-1,2,4-triazole-3-thiol are readily soluble in water and the aqueous solutions of these molecules were made using double distilled deionized water. 2-amino-2-thiazoline molecule is insoluble in water but freely soluble in methanol solution. The methanol is of spectroscopic grade and free from any impurities. In this case, Raman bands were normalized with respect to methanol solvent band. For all cases each reduction in concentration of the molecules, the addition of solvents in silver sol was kept constant. 2.4 Measurement of Absorption Spectra Surface plasmon oscillations within the metal have been established to be the principle contributor to surface enhancement of the Raman signal. The optimum Raman excitation frequency can be estimated if the plasmon adsorption maximum is known. The plasmon resonance frequency of the metal depends on the surface morphology [7, 8] and the surrounding medium [9]. From the absorption spectroscopic measurements of silver sol, one can get the plasmon absorption maximum of the silver particles. Shimadzu UV-VIS 2401PC spectrometer has been used to record the absorption spectra. A schematic optical diagram of the spectrometer is shown in Figure 2.1. The samples were taken in a 1 cm quartz cell. Figure2.1: Schematic diagram of the Spectrophotometer (UV-VIS 2401PC)

67 Fourier Transform Infrared Absorption Measurement Fourier Transform Infrared Absorption spectra were recorded on a Nicholet Magna 750 FTIR spectrophotometer. The schematic optical diagram of the spectrometer is shown in Figure 2.2. Figure 2.2: A schematic optical diagram of Nicholet Magna 750 FTIR spectrophotometer. The spectra were recorded in KBr pellet. About 1 mg of the sample and mg of potassium bromide are ground together finely, dried to remove moisture pressed at elevated temperature under high pressure into a small disc that measures about 1 cm in diameter and 1-2 mm in thickness. 2.6 Raman Spectra Measurement The necessary components for the observation of Raman spectra are: (a) source of monochromatic radiation, (b) an appropriate device to mount the sample illuminated optimally and efficient scattered light gathering mirrors, (c) a dispersive system and (d) a detection device. Raman scattering being a weak process as one out of a million incident photons in normal case, is expected to be Raman scattered, one needs an effective dispersive device with high light gathering optics and a very sensitive detection device. Single, double, double-pass tandem and triple

68 59 monochromators are now available as good dispersing system. Both photographic and photoelectric technique are used, PMT (photomultiplier tube) is still in common use. A very sensitive detection device, which has come up in last few years, is the CCD (charge-coupled device). In the present work, however we have used a Spex double monochromator (Model 1403) equipped with a cooled photomultiplier tube (Model R 928/5, Hamamatsu Photonics, Japan) and photon counting system. Samples were taken in quartz cell and were illuminated by different laser radiation available from a Spectra Physics Ar + laser (Model ). Spex Datamate 1B was used for monochromator control, data acquisition and analysis. A schematic diagram of the Raman set up is shown in Figure 2.3. Brief description of the instruments used is given below. Figure 2.3: A schematic diagram of the Laser Raman set up Spectra Physics Model Ar+ Laser The model Spectra Physics Ar + laser is a 5W CW laser. The laser head consists of a plasma tube closed at both ends by fused silica Brewster s angle windows, a solenoid, which provides necessary magnetic filed and an optical resonator. In the plasma tube, the optical cavity resonator is formed by a spherical reflector at the output end and a prism assisted by a flat mirror at the high reflector end. The flat mirror is used to select the wavelength. The resonator assembly is strongly held against quartz rods with springs. Aperture adjusting wheel is provided for changing the intra cavity aperture.

69 Sample Chamber (Illuminator) Figure 2.4: A schematic diagram of Spex 1403 double monochromator. Schematic diagram of the sample chamber is shown in Figure 2.4. The laser line is focused at the center of the 1 cm quartz cell (containing the samples) through its bottom face. The laser beam is focused to produce a beam of much smaller diameter, which extends over a short length before beginning to diverge again. The region in which the beam is concentrated is called focal cylinder. As the area of the focused beam, is about 10-3 times the area of the unfocused beam the irradiance at the sample is increased by about If high irradiance is harmful to the sample, focused beam is not used. The sample is placed in such a way that it is illuminated well and the scattered radiations are collected effectively. A lens is used to collect the scattered radiation for further dispensation. Additional concave mirrors M 3 and M 4 placed opposite to the monochromator side and on the top of the cell respectively are used to increase the observed intensity of the scattered light by 8 to 10 times. Filters and optical devices such as polarizer, analyzer, etc. may be inserted into the incident laser beam or in the scattered beam. The elliptical mirror M 5 and the plane mirror M 6 send the collected scattered light into the monochromator.

70 Spex Double Monochromator Model 1403 The Spex double monochromator (figure 2.4) covers a spectral range from cm -1 to cm -1 with an accuracy of ±1 cm -1 in range of cm -1. The spectral repeatability is ±0.2 cm -1 and the aperture is f/7.8. The holographic type grating in this instrument has 1800 grooves mm and it is mounted on a modified Czerny-Turner mount where the fundamental grating equation is M = d (sinα + sinβ) λ (2.1) Where, m=order; λ=wavelength; d=grating spacing; α=angle of incidence; β=angle of diffraction. For simplicity, equation (2.1) may be expressed as, M = 2d sinθ sinϕ Such that α = θ + ϕ and β = θ - ϕ; where θ = grating rotation angle measured from zero and ϕ = 10º, cos ϕ = (manufacturer s supplied values) Photomultiplier Tube In a Raman spectrometer, the photoelectric detection is achieved with a special kind of photocell called photomultiplier tube (PMT). The essential components of the PMT are a photosensitive cathode of low work function, and dynodes with the property to emit more secondary electrons that received in the incident electron bombardment. Photons falling on the photocathode, depending upon its quantum efficiency, get the electrons released from the cathode and these electrons are accelerated to different dynodes one after another, which results in the multiplication of number of electrons at every dynode. The total gain of a PMT may be represented as δ n, where δ the secondary gain and n is the number of dynodes. The electron multiplying process makes the PMT a suitable device for the detection of the lowlevel light signal. A schematic diagram of the PMT is given in Figure 2.5. Along with the signal electrons, there may be additional electrons randomly (thermally) emitted from the photocathode and dynodes. These electrons will also be multiplied and contribute as a noise to the main signal.

71 62 Figure 2.5: A schematic diagram of the photomultiplier tube Even with the no signal, due to these thermally emitted electrons in the PMT, the value of current or voltage developed constitutes the dark noise of the detecting device. The PMT is cooled by thermoelectric cryostat and circulating cold water at C cools the PMT housing. The dark current of a properly cooled PMT is quite small and therefore allows, the observation of even a very weak Raman bands. The PMT associated with the photon counting system in conjunction with a comprudrive and linear (in wave number) chart recorder, facilitates the recording of the Raman spectra. By properly cooling the PMT, selecting an appropriate time constant, and using a slow scan speed (1 cm -1 /sec), the Raman spectra with a reasonably good signal to noise ratio were recorded in the present work Data Acquisition System Spex Datamate 1B is a dedicated microcomputer, which performs the spectrometer control, data acquisition and analysis. The software has the provisions for background subtraction, integration, addition, division, selection of frequency range and intensity scaling, differentiation etc. Spectral data can be stored in a floppy disk. The storing facility could also be bypassed and real time spectra could be plotted directly by an X-Y plotter. The Datamate also supplies the high voltage required for

72 63 operating the photomultiplier tube with a stability of ± % after half an hour warm up. 2.7 Computational Software Ab initio and DFT calculation was performed by Gaussian 98 [10] and Gaussian 03 [11] software. This Gaussian is a connected system of programs for performing a variety of semi-empirical and ab initio molecular orbital (MO) calculations. Gaussian program is capable of predicting many properties of molecules and reactions, in the gas phase and in solution, including molecular energies and structures, vibrational frequencies, IR and Raman spectra, molecular orbitals etc. computations can be carried out on systems in their ground state or in excited state. The energies, structures and molecular orbitals can be predicted for periodic systems. Thus, Gaussian can serve as a powerful tool for exploring areas of chemical interest like substituent effects, reaction mechanisms, potential energy surfaces and excitation energies. In some cases the vibrational modes has been assigned in terms of Potential energy distribution (PED). The PED calculations were performed with the GAR2PED software [12] from Gaussian output files. Cartesian displacements of different vibrational modes have been displayed using the Molekel-4.2, win 32 package [13] and Gauss View 3.0 programs. Generalized two-dimensional correlation spectra are produced by 2Dshige software [14]. Shigeaki Morita in Professor Yukihiro Ozaki s group at Kwansei-Gakuin University develops 2Dshige. All the spectra reported in this thesis are produced using Microcal Origin version 6.0.

73 64 Reference: 1. L. H. Little, in Infrared Spectra of Adsorbed Species (Academic Press) New York, M. L. Hair, in Infrared Spectroscopy in Surface Chemistry New York, Electron Spectroscopy for Surface Analysis (Ed. H. Ibach) in Topics in Current Physics Vol. 4, Springer, Berlin, H. Ibach and D. L. Mills in Electron Energy Loss Spectroscopy and Surface Vibrations (Academic Press) New York, Vibrational Spectroscopy of Adsorbates, (Ed. R. F. Willis) in Springer Series in Chemical Physics, Vol. 17, Springer, Berlin, J. A. Creighton, C. G. Blatchford and M. G. Albrecht, J. Chem. Soc. Faraday Trans. II. 75, 790 (1979) 7. J. K. Sass, R. K. Sen, E. Meyer and H. Gerischer, Surf. Sci. 44, 515 (1974) 8. E. N. Economou, and K. L. Ngai, Adv. Chem. Phys. 27, 265 (1974) 9. R. H. Doremus, J. Appl. Phys. 35, 3456 (1964) 10. M. J. Frisch, G. W. Trucks, H. B. Schiegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, V. G. Zakrzewski, J. A. Jr. Montgomery, R. E. Stratmann, J. C. Burant, S. Dapprich, J. M. Millam, A. D. Daniels, K. N. Kudin, M. C. Strain, O. Farkas, J. Tomasi, V. Barone, M. Cossi, R. Cammi, B. Mennucci, C. Pomelli, C. Adamo, S. Clifford, J. Ochterski, G. A. Petersson, P. Y. Ayala, Q. Cui, K. Morokuma, D. K. Malick, A. D. Rabuck, K. Rahgavachari, J. B. Foresman, J. Cioslowski, J. V. Ortiz, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. Gomperts, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, C. Gonzalez, M. Challacombe, P. M. W. Gill, B. G. Johnson, W. Chen, M. W. Wong, J. L. Andres, M. Head- Gordon, E. S. Replogle, J. A. Pople, Gaussian 98; Gaussian, Inc.: Pittsburg, PA, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. A. Montgomery, Jr., T. Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G.

74 65 Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong, C. Gonzalez, and J. A. Pople, Gaussian, Inc., Pittsburgh PA, J. M. L. Martin, C. V. Alsenoy, GAR2PED, University of Antwerp, Molekel 4.2: P. Flukiger, H. P. Luthi, S. Portmann, J. Weber, Swiss centre for Scientific Computing, Manno (Switzerland) S. Morita, 2Dshige (c) Kwansei-Gakuin University,

75 66 CHAPTER 3 Adsorption of 2-Aminobenzothiazole on Colloidal Silver Particles: An Experimental and Theoretical Surface-Enhanced Raman Scattering Study 3.1 Introductory Remarks SERS is a well-established and highly effective technique of observing Raman scattering from species present at trace concentrations. The adsorptive site/sites and the orientation of the adsorbed molecule could be determined qualitatively by comparing the relative intensities and positions of the bands in the SERS with those in the NRS, of the pure or solvated analyte. Recently, quantum chemical ab initio and DFT calculations are successfully utilized to model the experimentally observed SERS spectra [1-3]. The compounds containing a thiazole ring have shown many useful biological properties. Benzothiazole and its derivatives are widely recognized as corrosion inhibitors and have been extensively used in the surface treatment of metals and alloys [4-7]. 2-aminobenzothiazole (2-ABT) molecule is known for their local anesthetic action and has numerous applications in human and veterinary medicine [8]. It is a metabolite of methabenzthiazuron and is reported to form the main fraction of soil bound residues. This chapter deals with the detail experimental and theoretical NRS, SERS and FTIR spectra along with tentative vibrational assignments of the observed bands of the biologically important 2-ABT molecule. The optimized structural parameters and the computed vibrational wavenumbers of the compound have been estimated from ab initio-hf and DFT calculations. Some vibrational modes of the molecule have been reassigned. The adsorptive behavior of 2-ABT on a colloidal silver surface at different adsorbate concentrations, close to that encountered under physiological conditions in living systems, has been elucidated from the SERS spectra. NRS spectra of the chemically prepared 2-ABT-Ag (I) complex and their comparison with the SERS spectra are also reported herein.

76 Results and Discussion 2-ABT was purchased from Aldrich Chemical Co. and was used without further purification. This molecule is readily soluble in acetonitrile (ACN) solution. The preparation of silver sol, samples, the detail experimental technique and a brief description of the used instruments are described in chapter 2. The process described by Creighton et al [9] was used to prepare the stable silver sol. The samples were taken in a quartz cell. The NRS in solution and the SERS measurements in sol were done by exciting the sample with nm radiation from a Spectra Physics Ar + ion laser at a power of 200 mw. For solid, laser power of 100 mw was used. Raman scattering was collected at right angle to the excitation. The FTIR spectrums of the powder samples were taken in KBr pellet using Nicolet Magna-IR 750 spectrometer series-ii Normal Raman and FTIR spectra of 2-ABT 2-ABT molecules has 16 atoms, hence it has 42 fundamental vibrations. It belongs to C s point group. Simple group theory predicts that 29 planar (A / species) and 13 non-planar (A // species) are expected to appear both in the Raman and in the IR spectra. The chemical structure of 2-ABT is shown in Figure 3.1. H16 y 15H 14H S 7 N N x H12 Figure 3.1: Schematic representation of the 2-ABT z H13 H11 Selected optimized structural parameters of free and adsorbed 2-ABT molecule calculated by DFT and RHF ab initio methods are listed in Table 3.1. The NRS of 2-ABT in 0.1M solutions and in neat solid are shown in panels a, and b respectively, of Figure 3.2. The panel c of the Figure 3.2 displays the calculated NRS spectrum.

77 68 Normalized intensity (a) 292 * * * (b) Counts/s can Raman activity (A O 4 /a.m.u) (c) Wavenumber/ cm Figure 3.2: Normal Raman spectra of 2-ABT (a) in 0.1 M solution (*denotes the solvent band) and (b) in solid state for λ exc = nm. The bottom spectra (c) the theoretical gas-phase Raman intensities calculated using RHF ab initio method The FTIR spectrum of the powdered sample in a KBr pellet is shown in Figure 3.3. Table 3.2 lists the FTIR, NRS and SERS band frequencies of the molecule along with their tentative assignments and probable Raman polarizability tensor elements. The observed and calculated vibrational frequencies of 2-ABT-Ag (I) complex are also shown in Table 3.2. In assigning the vibrational frequencies, literatures

78 69 concerning with the normal coordinate analysis and vibrational assignment of this [10] and of related molecules [11-14] have been consulted. The modes arising from the stretching and bending vibrations of the benzene and thiazole moieties of 2-ABT along with the scissoring mode of the externally attached -NH2 group are identified. An interesting observation can be drawn regarding assignment of the band centered at ~1015 cm -1. This band is intense and strongly polarized in NRS but appears as weak, but prominent in the FTIR spectra. It principally represents a planer mode and has now been assigned to the ring breathing (ν 1 ) vibration of the benzene moiety [15]. Previously this band was ascribed to the C- C-C trigonal bending [13]. There is a discrepancy in the assignment of another band centered at 747cm -1. This band is very strong in the FTIR spectra but weak in the NRS of solid. Figure 3.3: FTIR spectrum of 2-ABT of neat powder in KBr pellet. Generally, out-of-plane modes appear strongly in the infrared and weakly in the Raman [16-17] and so from this point of view its earlier assignment as C-H outof-bending is justified [13, 18, 19]. However, this band is moderately intense in the NRS of solution and is polarized. The band has been reassigned, and considering the frequency region, it is thought to arise from one of the C-C-C trigonal in-plane bend (ν 12 ) of the fused benzene moiety of 2-ABT molecule [15].

79 70 Table 3.1: Relevant structural parameters of 2-ABT and the changes therein upon binding to Ag at RHF and DFT level of theory RHF DFT 2-ABT 2-ABT-Ag Diff. 2-ABT 2-ABT-Ag Diff. Bond Lengths (Å) C 2 -C C 3 -C C 4 -S C 8 -S C 8 -N C 3 -N Interatomic Angles (degrees) C 2 -C 3 -C C 3 -C 4 -S C 4 -S 7 -C C 3 -N 9 -C S 7 -C 8 -N C 2 -C 3 -N C 1 -C 2 -C Dihedral Angle (degrees) C 5 -C 4 -C 3 -N Concentration-Dependent SERS spectra of 2-ABT Panels a-e of Figure 3.4 show the normalized SERS spectra of 2-ABT molecule at varied adsorbate concentrations in the range 1.0x10-3 M to 1.0x10-7 M. For each reduction of concentration of molecules in the silver sol, the concentration of acetonitrile (ACN) was kept constant, facilitating normalization of the SERS spectra. The normalization was done with respect to the 921 cm -1 band of ACN. It is established that ACN bands do not show any surface enhancement in the silver hydrosol [20] so any possible interaction of ACN with the silver colloid can be neglected.

80 Table 3.2: Observed and calculated Raman and IR bands of 2-ABT and 2-ABT-Ag(I) complex in varied environment and their tentative assignments* FTIR (obs) NRS Solid (obs) NRS Solid (calc) NRS soln SERS (10-4 M) Ag-complex (obs) Ag-complex (calc) Tentative assignment Probable tensor element Symmetry species RHF DFT RHF DFT 1644 vs NH 2 Scissors A / 1622 sh 1620 vvw vw 1610 ν(c=n)+nh 2 scissors α xx, α yy A / 1589 sh 1589 sh vvw 1588 vs ν(c=n)+nh 2 scissors α xx, α yy A / 1567 s vvw 1574 s ν(c=c) α yy A / 1529 vs 1539 s s P 1524 NH 2 scissors. A / 1443 vs 1452 ms s 1451 s ν(c=c) α xx, α yy A / 1309 ms 1315 ms vw ν(c-n) α zz,α xy A / 1282 ms 1286 vs s P 1288 vs ν(c-c) α xx, α yy A / 1245 ms 1251 vs s P 1249 vs ν(c-c) α xx, α yy A / 1124 s ms P 1128,1113 ms β(c H) α xx, α yy A / 1103 s vw N H deform A / 1064 sh 1066 ms ms 1070 w β(c H) α zz, α xy A / 1040 w ν(c C) α zz, α xy A / 1014 vw 1016 vs s P 1027vs, 1019 s Ring breathing; (ν 1 ) α xx, α yy A / ms 882 w γ(c H). α xz, α yz A // 741 vs 747 w ms P 748 vw 760 β(c C C); ν 12 α zz, α xy A / 713 vs 704 vs ms P 705 s ν(c S) α xx, α yy A /

81 72 Table 3.2 (Continued) FTIR NRS Solid NRS Solid NRS soln SERS Ag-complex Ag-complex Tentative assignment Probable Symmetry (obs) (obs) (calc) (10-4 M) (obs) (calc) tensor element species RHF DFT RHF DFT 685 w 681 ms ms w 653 ms ν(c S) α xx, α yy A / 502 vs s 500 ms β(c C C) α zz, α xy A / 476 ms 484 vs s k 392 s 405 β(c-c-c); ν 6b α xx, α yy A / 300 ms w 286 w γ(c-c-c) α yz, α xz A // 217 sh 217 ν(ag N) *β: in plane bend; ν : stretch, γ: out of plane bend, vs : very strong, s : strong, ms: medium strong, w: weak, vw: very weak, vvw: very very weak, sh :shoulder, P: polarized

82 73 Considering the entire concentration-dependent profile, we find that the concentration range between 1.0x10-3 M and 1.0x10-6 M is most sensitive not only for large signal counts but also for interesting variations in the relative intensities and band frequencies. nsity Nor malized inte Normalized intensity aliz ed inte nsity Norm (c) 211 (b) 215 (a) * * 380 * * * * * * * Normalized intensity Normalized intensity (e) (d) * * * * Wavenumber/cm * Wavenumber/cm -1 Figure 3.4: Normalized SERS spectra of 2-ABT in silver hydrosol at concentrations (a) 1.0 x 10-3 M, (b) 1.0 x 10-4 M, (c) 1.0 x 10-5 M, (d) 1.0 x 10-6 M, (e) 1.0 x 10-7 M for λ exc = nm (*denotes the solvent band). Significant band broadening are observed for the ring-breathing mode in the SERS spectra at 1.0x10-3 and 1.0x10-4 M adsorbate concentrations. It is characterized by the appearance of a blue shifted 1027 cm -1 mode along with 1019 cm -1 band. The concomitance of both these modes may indicate the existence of two types of adsorbed species namely M and N on the colloidal silver particles at these concentrations. The 1019 and 1027 cm -1 bands are ascribed to the breathing vibrations of benzene moiety of M and N species of 2-ABT respectively [22]. The adsorption of M species of 2-ABT, involves insignificant or no interaction of the benzene ring moiety in the adsorption process. N species, on the other hand, involves strong interaction of the benzene ring in the adsorption mechanism. With

83 74 lowering in concentration, the 1027 cm -1 band, gains in intensity and at 1.0x10-6 M, it is further blue shifted and appears as single well-resolved intense band at 1031 cm -1 with the disappearance of 1019 cm -1 mode. Figure 3.5: Bar diagram indicating FWHM of the ring breathing (1019 cm -1 i.e.ν 12 ) mode in NRS and in SERS. The disappearance of 1019 cm -1 band in the SERS spectra at 1.0x10-6 M can be substantiated from the Figure 3.5, represents a bar diagram indicating FWHM of the ring-breathing mode in NRS and in SERS at varied adsorbate concentrations. A conspicuous increase in bandwidth of the ring-breathing vibration in the SERS spectrum between 1.0x10-3 and 1.0x10-5 M with respect to its NRS counterpart in solution is observed. The FWHM of this mode is found to drop substantially at 1.0x10-6 M, where it becomes almost comparable with the NRS. However, significant 16 cm -1 blue shift of the ring breathing in the SERS spectra in comparison with the corresponding NRS of solution, together with the disappearance of 1019 cm -1 band may indicate considerable population density of N type of adsorbed species at 1.0x10-6 M concentration. The blue shift of the ring-breathing mode as commonly observed in biomolecules [22-25] can be attributed to redistribution of electronic charge density in

84 75 the ring because of adsorption [26]. Involvement of the fused benzene ring in the adsorption process with the lowering in concentration can be further substantiated by the increase in intensity of 394 and 1574 cm -1 bands, assigned to C-C-C in-plane bending and C=C stretching respectively. In fact 1574 cm -1 band is also blue shifted approximately ~12 cm -1 compared to its NRS counterpart. Apart from the interaction of the benzene ring of 2-ABT in the adsorption process, the fused thiazole moiety of the molecule can also bind to the silver surface through the lone pair electrons of either nitrogen or sulfur atom or through both of them. More favored adsorptive site can be enumerated theoretically by estimating the partial atomic charges on each of these probable active sites [16, 22]. The higher the negative charge density on the atom, the higher is the probability of it acting as an adsorptive site for the silver substrate. Theoretical results estimated from DFT/RHF ab initio calculations shows that the partial atomic charges on nitrogen and sulfur atoms determined by the Natural Population Analysis (NPA) are / and / respectively. The negative charge density is thus observed to be more appreciable on nitrogen than on sulfur atom, thereby indicating the active involvement of nitrogen atom in the adsorption process. The appearance of shoulder at around 217 cm -1 in the concentration dependent SERS spectral profile, ascribed to Ag-N stretching vibration [1, 27-29] indicates that the fused thiazole moiety of 2- ABT molecule is indeed adsorbed through the ring nitrogen atom. Interesting observation can be drawn regarding 706 cm -1 band present in the entire concentration dependent SERS spectra. This band, ascribed to C-S stretching vibration [13, 19] exhibits essentially the same frequencies and band shapes in the adsorbed and bulk phase environments. This evidence further substantiates insignificant involvement of sulfur atom in the adsorption process. The lone pair electrons on the nitrogen atom of the externally attached NH 2 group can also be the possible active site in the adsorption mechanism. The partial atomic charge on this nitrogen atom determined by NPA according to DFT and RHF ab initio calculation is and respectively. In the concentration dependent SERS spectra, we observed considerable enhancement and variation in intensity of bands around 1550, 1588 and 1620 cm -1 ascribed to NH 2 scissoring mode. This may signify some interaction of the nitrogen atom of NH 2 group with the silver surface. However, among these vibrations, 1588 and 1620 cm -1 bands are

85 76 known to be overlapped with C=N stretching mode of the thiazole moiety of 2-ABT [11-13]. So the enhancement and intensity variation of these modes may be due to the resultant interaction involving nitrogen atom of the NH 2 group and thiazole moiety of 2-ABT molecule. The N-H stretching modes as often reported in cm -1 wave number range [15,22] are however not observed in the concentration dependent SERS spectra, probably due to the intense broad background of OH stretching mode (centered around 3400 cm -1 ) of bulk water. The N-H stretching vibration may therefore be completely masked by this intense broad background and hence could not be identified separately. The involvement of both the endocyclic and exocyclic nitrogen atoms in the adsorption process may result in the appearance of a broad shoulder in the SERS spectra at ~217 cm -1. The relative enhancement of the Raman bands of 2-ABT on the adsorbate concentration has been estimated. Figure 3.6 shows the variation of the normalized SERS signal intensities of 703, 1019, 1573 and 1583 cm -1 bands with the logarithm of concentration. It is observed that the SERS signal for two pairs of bands at 703, 1583 cm -1 and at 1019, 1573 cm -1 increase in intensity as the concentration of the adsorbate molecule is lowered; attain a maximum at 1.0x10-4 M and 1.0x10-6 M respectively. The former pair of bands at 703 and 1583 cm -1 represents vibrational signatures principally contributing from the thiazole ring while the later pair at 1019 and 1573 cm -1 represents the contribution from the benzene ring of 2-ABT. It is now well established that, on silver island films [30] and on silver colloids [16, 31], maximum enhancement is observed when a monolayer of adsorbate molecule is formed on the surface, and that, as multilayers are formed, the SERS signal decreases. It therefore seems plausible that the monolayer of the adsorbed 2-ABT is formed on the colloidal silver surface at two different adsorbate concentrations, which show maximum enhancement of the SERS signal Orientation of the 2-ABT Molecule on the Silver Surface In order to have a precise idea regarding the orientation of 2-ABT at different adsorbate concentrations, the apparent enhancement factors (AEF) of some selected Raman bands is estimated using the relation we used before [16, 22, 31]. Accordingly AEF = σ SERS [C NRS ] / σ NRS [C SERS ] (3.1)

86 77 C and σ represent the concentration and the peak area of the Raman bands measured from baseline. They are shown in Table-3.3. Orientation of the molecule has been estimated following the surface selection rule, as predicted by Moskovits [32] and Creighton [33]. According to this rule, those vibrations having larger component of polarizability in the direction normal to the surface will be enhanced more. Table 3.3: Apparent enhancement factors of some selected Raman bands of 2ABT NRS of solution Apparent enhancement factors at various concentrations (cm -1 ) 1.0x10-3 M 1.0x10-4 M 1.0x10-5 M 1.0x10-6 M 1.0x10-7 M x x x x x x x x x x x x x x x x x10 6. Let us consider x and y correspond to the long and short axis of the molecule and z is perpendicular to the molecular plane. If 2-ABT molecule is considered to be lying in the xy plane, then for edge-on adsorption, the vibration of the in-plane A / species transforming as yy (when short axis of the molecule is vertical to the surface) or xx (when long axis of the molecule is vertical to the surface) is expected to undergo significant enhancement. The least intense band should belong to the out-of-plane A // species transforming as yz and xz. A moderate 3-4 orders of enhancement of all the bands, as clearly seen from Table 3.3, principally representing in-plane vibrations of the A / species at an adsorbate concentration of 1.0x10-4 M. The enhancement factor increases by 1-3 orders at 1.0x10-6 M concentration. No significant enhancement contributing from the out-of-plane vibrations of the A // species of the molecule are recorded. Moreover, other in-plane modes at around 392, 1588 and 1620 cm -1, whose exact enhancement could not be predicted, are also significantly enhanced. These results together with the appearance of Ag-N stretching vibration suggest that 2-ABT molecules are adsorbed onto the metal surface through the nitrogen atoms of the thiazole moiety and also possibly through the externally attached amino group, with the molecular plane nearly vertical to the surface at all adsorbate concentrations.

87 78 The M-type of species that are prevalent between 1.0x x10-5 M adsorbate concentrations are thought to be adsorbed vertically in such a way so that the x-axis (long axis) of the molecule remains nearly vertical to the silver surface. This type of adsorption geometry precludes any direct interaction of the benzene ring moiety with the silver surface. The N-type species that co-exist at 1.0x x10-5 M concentration and predominantly exist at 1.0x10-6 M are probably adsorbed vertically with the y-axis (the short axis) of the molecule almost normal to the silver surface. This type of adsorption favors the direct involvement of benzene ring moiety with the silver surface. Figure 3.6: Concentration dependence of the intensity of (a) 707, (b) 1587, (c) 1573 (d) 1020 cm -1 SERS bands of 2-ABT. Different forms of vertical adsorption geometry of the M and N species and their relative population in the colloidal silver surface may result in monolayer coverage of 2-ABT molecule at two different adsorbate concentrations as observed in Figure 3.6.

88 NRS of the 2-ABT-Ag Complex Figure 3.7(a) shows the NRS of 2-ABT-Ag (I) complex. Well-resolved Raman bands whose vibrational signatures resemble very well with the SERS spectrum of the molecule characterize the Raman spectrum of the complex. Ag-N stretching vibration at 215 cm -1 is well identified, indicating considerable metal-adsorbate interaction. Interesting conclusion can be drawn regarding the appearance of ring breathing mode at around 1019 cm -1. Intenc ity (a.u.) 2abthcomplex (a) Raman activity ( A 04 /a.m.u. ) Wavenumber/cm (b) Figure 3.7: Normal Raman Spectra of (a) 2-ABT-Ag(I) complex in neat solid for λ exc = nm. The bottom spectrum (b) is the theoretical gas-phase Raman intensities of 2ABT- Ag (I) model calculated using RHF ab initio method. The unique appearance of this band suggests insignificant interaction of the benzene ring moiety in the adsorption process. Thus, the binding arrangement of 2- ABT-Ag (I) complex closely mimics with the M -type adsorbed species of the SERS spectrum. The theoretical gas-phase Raman spectrum of modeled 2-ABT-Ag complex is shown in Figure 3.7(b). The optimized geometries of modeled 2-ABT-Ag (I) complex obtained from DFT and RHF ab initio level of theory are shown in Figure 3.8(a) and (b) respectively.

89 80 As one can see from the Figure 3.7(a) and (b) and from Table 3.2, that theoretically predicted frequencies almost matches with the experimental results within tolerable limit. DFT RHF a b Figure 3.8: Optimized geometritics of modeled 2-ABT-Ag (I) complex obtained from (a) DFT (b) RHF ab initio level of theory. This immediately allows us to investigate the changes in the structural parameters of the 2-ABT molecule because of Ag interaction. They are listed in Table-3.1. These parameters may be closely related to the molecular geometry of the M -type adsorbed species of 2-ABT molecule. 3.3 Conclusion The adsorption behaviors of biologically significant 2-Aminobenzothiazole molecules on colloidal silver particles have been investigated by SERS aided by density functional theory and ab initio Restricted Hatree-Fock computation of vibrational frequencies of the isolated molecule. The optimized structural parameters of free and modeled adsorbed molecule have been estimated from the abovementioned level of theory. Some vibrational modes of the free molecule have been reassigned. Existences of two types of adsorbed species have been inferred from the concentration dependent SERS spectral profile. The orientations of the two adsorbed species have been estimated from the surface selection rule. The Raman vibrational bands of one type of adsorbed species closely resemble with the NRS of chemically prepared and theoretically modeled of 2-ABT-Ag (I) co-ordination compound.

90 81 Reference: 1. R. F. Aroca, R. E. Clavijo, M. D. Halls, H. B. Schlegel, J.Phys.Chem. A 104, 9500, (2000) 2. M. Bolboaca, T. Iliescu, Cs. Paizs, F. D. Irimie, W. Kiefer, J.Phys.Chem. A 107, 1811, (2003) 3. M. Baia, L. Baia, W. Kiefer, J. Popp, J.Phys.Chem. B 108, 17491, (2004) 4. G. Schimitt, Brit. Corrosion J. 19, 165, (1984) 5. M. Musiani, G. Mensoli, M. Fleischmann and R. B. Lowry, J. Electroanal. Chem. 217, 187, (1987) 6. J. C. Marconato, L. O. Bulhoes, M. L. Temperini, Electrochim Acta 43, 771, (1998) 7. M. Obsawa, W. Suetaka, Corros. Science 19, 709, (1979) 8. J. V. N. Vara-Prasad, A. Panapoulous, J. R. Rubin, Tetrahedron Lett. 41, 4065, (2000) 9. J. A. Creighton, C. G. Blatchford, M. G. Albrecht, J. Chem. Soc., Faraday Trans. 275, 790, (1979) 10. K. Koglin, E. G. Witte, R. J. Meier, Vib. Spectrosc. 33, 49, (2003) 11. W. B. Collier, T. D. Klots, Spectrochim Acta A 51, 1255,(1995) 12. T. D. Klots, W. B. Collier, Spectrochim Acta A 51, 1273,(1995) 13. S. Mohan, A. R. Prabakar, S. Prameela, Ind.J.pure & Appl. Phys. 29, 672, (1999) 14. B. Pergolese, A. Bigotto J. Raman spectrosc. 34, 84, (2003) 15. G. Varsanyi, In Vibrational spectra of benzene derivatives; Academic Press: New York and London, (1969) 16. J. Chowdhury, M. Ghosh, T. N. Misra, Spectochim. Acta A 56, 2107, (2000) 17. S. C. Wait, J. C. Mcnerney, J.Molec. Spectrosc. 34, 56, (1970) 18. B. Pergolese, A. Bigotto, J. Raman Spectrosc. 33, 646, (2002)

91 N. Sandhyarani, G. Skanth, S. Berchmans, V. Yegnaraman, T. Pradeep, J. Colloid and Interface Sci. 209, 154, (1999) 20. J. Chowdhury, M. Ghosh J. Colloid Interface Sci., 277, 121, (2004) 21. P. Gao, M. J. Weaver, J. Phys. Chem. 89, 5040, (1985) 22. J. Chowdhury, K. M. Mukherjee, T. N. Misra, J. Raman Spectroscopy, 31, 427, (2000) 23. B. Giese, D. McNaughton, J. Phys. Chem. B 106, 101, (2002) 24. S. Sanchez-Cortes, J. V. Garcia-Ramos, Vib. Spect. 4, 185, (1993) 25. B. Giese, D. McNaughton, Phys. Chem. Chem. Phys. 4, 5171, (2002) 26. L. E. Comfeita, S. Sanchez-Cortes, J. V. Garcia-Ramos, J. Raman Spectrosc. 26, 149, (1995) 27. D. W. Boo, M. S. Kim, K. Kim, Bull Korean Chem. Soc. 9, 311, (1988) 28. M. Miranda, J. Raman Spectrosc. 28, 205, (1997) 29. M. Miranda, Chem. Phys. Lett. 340, 437, (2001) 30. P. N. Sanda, J. M. Warlaumont, J. E. Dermuth, J. C. Tsang. T. Christmann, J.A. Bradley, Phys. Rev. Lett. 45, 1519, (1980) 31. J. Chowdhury, M. Ghosh, T. N. Misra, J. Colloid and Interface Sci. 228, 372, (2000) 32. M. Moskovits, J. Chem Phys. 77, 4408, (1982) 33. J. A. Creighton, Sur. Sci. 124, 209, (1983)

92 83 CHAPTER 4 Experimental and Theoretical Surface Enhanced Raman Scattering Study of 2-Amino-4- methylbenzothiazole Adsorbed on Colloidal Silver Particles 4.1 Introductory Remarks Useful information regarding the nature and orientation of adsorbed molecular species and the adsorbate metal interaction mechanisms have established using extensive experimental [1, 2] and theoretical [3-6] SERS technique. Organic compounds containing sulfur have shown to offer a high degree of inhibition to the corrosion of metals in acidic media [7]. Benzothiazole and its derivatives are extensively used in the surface treatment of materials, particularly in the field of anticorrosion protection of metals and alloys [8-10]. Apart from their inherent anticorrosion properties, benzothiazoles also form an important class of chemical species, which are involved in numerous applications, including human and veterinary medicine. Considering the enormous industrial and biological importance, this chapter presents the detailed experimental and theoretical NRS, SERS and FTIR spectra of 2- Amino-4-Methyl Benzothiazole (2-AMBT) molecule. The adsorptive behavior of 2- AMBT on the colloidal silver surface at two different adsorbate concentrations recorded in different time domains has been elucidated from the SERS spectra. The silver surface may serve as an analogue for the artificial biological interface. The experimentally observed SERS spectra are compared with the theoretically modeled 2-AMBT-Ag (I) surface complexes using ab initio RHF and DFT calculations. The most favorable adsorptive sites of the 2-AMBT molecule have been estimated by natural population analysis (NPA) using the above-mentioned high level of theories. 4.2 Results and Discussion 2-ABT molecule was purchased from Aldrich Chemical Co. and was used without further purification. The preparation of silver sol, samples, the detail

93 84 experimental technique and brief description of the used instruments are described in chapter 2 and Normal Raman and FTIR spectra of 2-AMBT and their vibrational assignment 2-AMBT molecule has 19 atoms; hence, it has 51 fundamental vibrations. It belongs to C s point group and consequently, 35 planar (A / species) and 16 nonplanar (A // species) fundamental vibrations are expected to appear both in the Raman and in the FTIR spectra. The optimized structure of 2-AMBT is shown in Figure 4.1. Figure 4.1: The optimized structure of 2-amino-4-methylbenzothiazole obtained from BPW91/6-31G (d, p) level of theory Selected optimized structural parameters of free and adsorbed 2-AMBT molecule calculated by DFT and RHF ab initio methods are listed in Table 4.1. The carbon (C 16 ) atom of the exocyclic CH 3 group of 2-AMBT is sp 3 hybridized with the relevant bond angle ~ The normal Raman spectra of 2-AMBT at 0.1M solution

94 85 and in neat solid are shown in Figures 4.2(a) and 2(b) respectively. The calculated NRS spectrum is shown in Figure 2(c). Table-4.1: Relevant Structural Parameters of 2-AMBT and Model-III Ag complex of 2-AMBT molecule calculated from RHF/6-31G (d, p) and BPW91/6-31G (d, p) and RHF/Lanl2DZ, BPW91/Lanl2DZ levels of theories respectively. RHF DFT 2-AMBT MODEL-III 2-AMBT MODEL-III Bond Lengths (A 0 ) C 2 -C C 3 -C C 4 -S C 8 -N C 8 -N C 3 -N Interatomic Angles (degrees) C 2 -C 3 -C C 3 -C 4 -S C 4 -S 7 -C N 9 -C 8 -N S 7 -C 8 -N H 17 -C 16 -H Dihedral Angles (degrees) C 5 -C 4 -S 7 -C C 5 -C 4 -C 3 -N It is to be emphasized that the calculated Raman spectrum represents the vibrational signatures of molecules in its gas phase. Hence, the experimentally observed Raman spectrum of solid and in solution may differ significantly from the calculated spectrum [11]. However, one can see that there is a substantial agreement regarding the Raman intensities as well as the position of the of the peaks between the experimental and the calculated spectra [12-14] The observed FTIR spectrum of the powdered sample in a KBr pellet is shown in Figure-4.3. Table 4.2 lists the FTIR, NRS, SERS band frequencies of the molecule along with their tentative assignments and probable Raman polarizabilty tensor elements. The modes arising from the stretching and bending vibrations of the benzene and thiazole moieties of 2-AMBT along with the scissoring, rocking and wagging modes of the externally attached NH 2 and -CH 3 groups are identified. In assigning the vibrational frequencies, literatures concerning with the normal coordinate analysis and vibrational assignments of the related molecules [15-19] have been consulted.

95 86 Normalized intensity (a) * * * Counts/Scan (b) Raman activity (A 0 30 (C) Wavenumber/cm -1 Figure 4.2: Normal Raman spectra of 2-AMBT (a) in 0.1 M solution (asterisk denotes the solvent band) and (b) in solid state for λ exc = nm. The bottom spectra (c) the theoretical gas-phase Raman spectrum calculated using RHF ab initio method Absorbance Wavenumber/cm -1 Figure 4.3: FTIR spectrum of 2-AMBT of neat powder in KBr pellet.

96 87 Table 4.2: Observed and calculated Raman, IR and SERS bands of 2-AMBT in varied environments and their tentative assignments FTIR (obs) NRS Solid (obs) NRS Solid (calc) NRS soln SERS (10-5 M) RHF DFT Tentative assignment Probable tensor element Symmetry species 1629 vs 1635 vw vw 1620 s ν(c=n)+nh 2 Scissors α xx A / 1586 w 1581 s vw 1580 m ν(c=c)+nh 2 scissors α xx A / ν(c=c)+nh 2 scissors α xx A / 1526 vs 1510 vs s 1555 s ν(c=n)+nh 2 scissors α xx A / 1473 w β(c H) α xx A / CH 3 osc 1454 s 1452 vw m 1446 m ν(c=c); CH 3 osc. A A / / 1432 vvw 1411 m 1411 w m β(c H);ν(C N); CH 3 osc vw 1371 vw CH 3 osc A A / / 1321 m 1311 w ν(c=c) A / C NH 2 bending.(i.p.) A / 1272 m 1274 vs vs 1273 s ν(c N); β(c H) α xx A / 1246 w 1243 m m β(c H), C CH 3 stretching. α xx A /

97 88 Table 4.2 (Continued) FTIR (obs) NRS Solid (obs) NRS Solid (calc) NRS soln SERS (10-5 M) RHF DFT Tentative assignment Probable tensor element Symmetry species 1185 m m 1180 s β(c H) α xx A / 1156 w vw 1140 vs β(c H) α xx A / 1110 m 1116 w β(c H) α xx A / 991 vw 998 w vvw 999 m Ring breathing A / 895 w 893 vw γ(c H) // A 872 w s Ring deformation α xx A / γ(c H) // A Ring deformation α xx A / 789 m 763 s A / 741 m m γ(c H) // A o.o.p. ring deformation. A // 690 w A / 673 m w 672 w α (C C C) α xx A / ν (C S) C NH 2 bending (o.o.p.) // A 565 vw 565 m m 575 m NH 2 wagging α yz, α xz A // o.o.p. ring deformation A // 527 m m 519 m Ring deformation; C CH 3 bending / A

98 89 Table 4.2 (Continued) FTIR (obs) NRS Solid (obs) NRS Solid (calc) NRS soln SERS (10-5 M) Tentative assignment Probable tensor element Symmetry species RHF DFT NH 2 wagging α yz, α xz A // NH 2 wagging; Ring bending (o.o.p.) // A 469 m vvw 458 w α(c C C) A/ 353 w NH2 twist (o.o.p.) A// 332 w vw NH2 twist (o.o.p.) A// NH2 twist (o.o.p.) A// 241 m vvw Ring bending (o.o.p.) A// 217 sh ν(ag N) C CH3 bending.(i.p.) A/ Ring bending (o.o.p.) A// Ring bending (o.o.p.) A// CH3 osc A/

99 SERS Spectra of 2-AMBT Scanning through these spectra (Figure-4.4 and 4.5) immediately reveal that the prominent SERS bands at 1.0 x 10-4 M and 1.0 x 10-5 M adsorbate concentrations principally represent the in-plane vibrational modes of the benzene and the thiazole moieties of 2-AMBT molecule. The in-plane ring deformation modes at 393 cm -1 and 860 cm -1 of the benzene moiety of the molecule are significantly enhanced. Significant enhancements are observed for the bands centered at ~1001 cm -1 and at 1274, 1580 cm -1 assigned to the breathing and stretching vibrations respectively of the benzene ring moiety of 2-AMBT molecule. The in-plane C-H bending modes at ~ 1140 cm -1 and 1176 cm -1 are also considerably enhanced. Interestingly, we observe that the vibrational signatures principally contributing from the co-ordinates of the benzene ring moiety of the molecule in the SERS spectra exhibit the same band shapes and show very small or no shift compared to its NRS counterpart in solution. These observations suggest weaker or no interaction with the π-electron cloud of the benzene ring moiety of 2-AMBT molecule in the adsorption process. The fused thiazole moiety of the molecule, however, can bind to the colloidal silver surface through the lone pair electrons of either the nitrogen (N 9 ) or sulfur (S 7 ) atom or through both of them. The best way of treating this problem is to enumerate the negative charge density on each of these probable active sites [20-21]. The higher is the negative charge density on the atom, the higher is the probability of it to act as an adsorptive site for the silver substrate. Theoretical results estimated from DFT/RHF ab initio calculations shows that the partial charges on the N 9 and S 7 atoms determined by the natural population analysis (NPA) are / and / respectively. The negative charge density is thus observed to be more appreciable on the nitrogen (N 9 ) atom than on sulfur (S 7 ) atom, thereby indicating the active involvement of the nitrogen (N 9 ) atom in the adsorption process. The appearance of shoulder ~ 217 cm -1, assigned to Ag-N stretching vibration [11, 22-24] in the SERS spectral profile indicates that the fused thiazole moiety is indeed adsorbed on the colloidal silver surface through the ring nitrogen (N 9 ) atom via σ- bond formation. Apart from the N 9 atom of the thiazole moiety of the molecule, the lone pair electrons on the nitrogen (N 10 ) atom of the exocyclic NH 2 group can also take part in the adsorption process of 2-AMBT molecule. The partial atomic charge on this

100 91 nitrogen atom determined by NPA according to DFT and RHF ab initio calculation is and respectively. Normalized intensity (a) 217 * * * Wavenumber/cm -1 Normalized intensity (b) 400 * * * Wavenumber/cm -1 Figure 4.4: Normalized SERS spectra of 2-AMBT in silver hydrosol at concentrations (a) 1.0 x 10-4 M and (b) 1.0 x 10-5 M for freshly prepared colloid-molecule mixture. (λ exc = nm, asterisk denotes the solvent band).

101 92 Normalized intensity 2 1 (a) * * * 1620 Normalized intensity (b) * * * Raman activity (A 0 /a.m.u.) (C) Wavenumber/cm -1 Figure 4.5: Normalized SERS spectra of 2-AMBT in silver hydrosol at concentrations (a) 1.0x10-4 M and (b) 1.0x10-5 M for 3 days aged colloid-molecule mixture. (λ exc = nm, asterisk denotes the solvent band). The bottom spectrum (c) is the theoretical gas-phase Raman spectrum of 2-AMBT-Ag (I) Model-III complex calculated using RHF ab initio method.

102 cm -1 (1555cm -1 ) 1591cm -1 (1580 cm -1 ) 1627 cm -1 (1620cm -1 ) Figure 4.6: Cartesian displacement and calculated (BPW91/6-31G (d, p)) vibrational modes of 2-AMBT; The numbers in the parentheses referred to the experimental value of the assigned band The SERS spectra recorded in different time domains at varied adsorbate concentrations show considerable enhancement and variation in intensity of the bands around 1552 and 1618 cm -1 ascribed to the coupled vibrations involving NH 2 scissoring and C=N stretching modes. In fact, the 1552 cm -1 band is blue-shifted by ~ 21 cm -1 in comparison with its NRS counterpart in solution. The Cartesian displacements and normal modes of vibrations involving NH 2 group calculated from DFT calculation are shown in Figure 4.6. However, the enhancements and intensity variations of 1552 and 1632 cm -1 bands along with the appreciable blue shift of 1552 cm -1 mode in the SERS spectra may be due to the resultant interaction of the NH 2 group and the thiazole moiety of the 2-AMBT molecule. The involvement of both the endocyclic and exocyclic nitrogen atoms in the adsorption process may result in the appearance of a broad shoulder in the SERS spectra ~ 217 cm -1. Thus, in general the SERS spectral analysis has been utilized to refine the vibrational analysis of the benzene and thiazole moieties of 2-AMBT molecule.

103 Orientation of the 2-AMBT Molecule on the Silver Surface In order to have a precise idea regarding the orientation of 2AMBT at different adsorbate concentrations, we estimate the apparent enhancement factors (AEF) of some selected Raman bands using the relation [21, 25]. Accordingly AEF = σ SERS [C NRS ] / σ NRS [C SERS ] (4.1) Where C and σ represent the concentration and the peak area of the Raman bands measured from baseline. Let us consider x and y correspond to the long and short axis of the molecule and z is perpendicular to the molecular plane. If 2-AMBT molecule is considering to be lying in the xy plane then for the edge-on adsorption, the vibration of the in-plane A / species spanning as xx (where long axis of the molecule is vertical to the surface) or yy (when the short axis of the molecule is vertical to the surface) is expected to undergo significant enhancement. Table-4.3 Apparent enhancement factors of some selected Raman bands of 2-AMBT at varied concentrations recorded in different time domain. NRS of Solution (cm -1 ) Fresh colloidmolecule mixture Apparent Enhancement Factor at various concentrations 1.0 x 10-4 M 1.0 x 10-5 M Aged colloidmolecule Fresh colloid- mixture molecule mixture Aged colloidmolecule mixture x x x x x x x x x x x x x x x x x x x 10 5 The least intense band should belong to the out-of-plane A // species spanning as yz and xz. It is clearly seen from Table 4.3 that we obtain a moderate 3-5 orders of magnitude enhancement of all the bands principally representing the in-plane vibrations of the A / species of 2-AMBT molecule at 1.0 x 10-4 M and 1.0 x 10-5 M adsorbate concentrations for freshly prepared and aged colloid-molecule mixture. Moreover, the other in-plane modes at around 398 and 860 cm -1, whose exact enhancement could not be predicted, are also significantly enhanced.

104 95 Figure-4.7 shows the bar-diagram indicating the logarithm of Apparent Enhancement Factor for the selected SERS band at (I) 1.0 x 10-4 M and (II) 1.0 x 10-5 M concentrations for freshly prepared and aged colloid-molecule mixture. Log (AEF) I 563 cm -1 A F 1184 cm cm cm -1 Log (AEF) II 563 cm -1 A F 1184 cm cm cm Wavenumber (cm -1 ) Wavenumber (cm -1 ) Figure 4.7: Bar diagram indicating the logarithm of Apparent Enhancement Factor for the selected SERS band at (I) 1.0 x 10-4 M and (II) 1.0 x 10-5 M concentrations. (F and A signify the AEF of freshly prepared and aged colloid-molecule mixture). Interesting interpretation can be drawn regarding the enhancement of the vibrational signatures contributing from the exocyclic NH 2 group.sers spectra, recorded in different time domains, at the above-mentioned adsorbate concentrations show significant enhancement of ~4 orders for the band centered at ~ 574 cm -1, ascribed to the out-of-plane NH 2 wagging mode. This band is considerably blueshifted (~11 cm -1 ) in comparison to its NRS counterpart in solution. This substantiates our earlier prediction about the involvement of N 10 atom in the adsorption process. These results together with the appearance of the Ag-N stretching vibration suggest that 2-AMBT molecules are adsorbed onto the metal surface through the nitrogen (N 9 and N 10 ) atoms of the thiazole moiety. And also the adsorption is possibly through the externally attached NH 2 group with the molecular plane nearly vertical to the surface at 1.0 x 10 4 M and 1.0 x 10-5 M adsorbate concentrations recorded in different time domains. As the benzene ring moiety of the molecule is not involved in the adsorption process, the 2-AMBT molecule is probably adsorbed vertically in such a way that the x-axis (long axis) of the molecule remains nearly vertical to the silver surface. This type of adsorption geometry precludes any direct interaction of the benzene ring moiety with the silver surface. However, the appearance and enhancement of both the in-plane and out-of-plane modes contributing from the NH 2 group dictates us to believe that N-H legs of the

105 96 externally attached NH 2 group are neither flat nor perpendicular to the surface but are tilted. The relative small variation in intensity and enhancement factor of the bands contributing from NH 2 scissoring and N-H out-of-plane modes at 1.0 x 10 4 M and 1.0 x 10-5 M adsorbate concentrations in different time domains may signify fluxional motion of the amino group on the colloidal silver surface DFT and ab Initio Calculations on Models of Surface Complexes The calculations of model surface complexes constituted by molecules bound to metal atoms provide useful information about the interaction between the adsorbate and metal substrate [26-28]. The DFT calculations on three different model complexes have been carried out. The first one having a silver atom bound to the nitrogen (N 9 ) atom of the thiazole moiety (model-i). The second one consisting of one silver atom bound to both the nitrogen atoms (N 9 and N 10 ) of the thiazole moiety and the exocyclic NH 2 group (model-ii). The third one comprising of two individual silver atoms, one bound to the nitrogen (N 9 ) atom of the thiazole moiety of 2-AMBT molecule and the other one bound to the nitrogen (N 10 ) atom of the externally attached NH 2 group (model-iii). Ab initio RHF calculations for model-iii complex are also reported. The main aim of these type calculations is to identify the species that really adsorbed on silver surface by the best agreement with the SERS bands. Table-4.4 shows the calculated frequencies of three probable model complexes along with the experimentally observed SERS bands at 1.0 x 10-4 M and 1.0 x 10-5 M adsorbate concentrations. As one can see from Table-4.4 and from Figure-4.5(c), the theoretically predicted frequencies for Model-III complex almost match the experimental results within a tolerable limit. Moreover, the DFT results suggest that thermodynamically Model-III is most stable in the ground state, it being ~ ev and ~ ev, more stable than Model-I and Model-II respectively. These results also indicate that the binding arrangement of 2-AMBT-Ag (I) closely resembles the theoretically modeled Model-III complex.

106 97 Table 4.4: Calculated frequencies of three probable model complexes along with the experimentally observed SERS bands at 1.0x10-4 M and 1.0x10-5 M adsorbate concentrations Observed SERS Bands (cm -1 ) at different Concentrations MODEL-I MODEL-II MODEL-III 10-4 M 10-5 M DFT DFT RHF DFT

107 Electronic Absorption Spectra of Silver Colloid with Added 2-AMBT Figure 4.8 (I) shows the room temperature UV-visible electronic absorption spectra of Ag sol, as prepared, before and after the addition of 2-AMBT molecule at 1.0 x 10-4 M adsorbate concentration recorded in different time domains. The pure stable silver sol shows a single extinction maximum at 392 nm. When 1.0 x 10-4 M 2- AMBT is added to the sol, the extinction maximum at 392 nm diminishes with the appearance of a broad hump at around ~ 565 nm nm (a) (I) Absorbance 2.5 (c) (b) 565 nm 665 nm wavelength (nm) Counts/Mw/Scans a) 860cm -1 b) 1620cm -1 c) 1140cm -1 d) 1580cm -1 d c b a (II) wavelength (nm) Figure 4.8: (I) Room temperature UV-visible absorption spectra of silver sol (a) pure sol and with added 2-AMBT for (b) freshly prepared (c) 3-days aged colloid-molecule mixture. The concentration of 2-AMBT in silver sol is kept at 1.0 x 10-4 M. (II) Excitation wavelength dependence of (a) 860 cm -1 (b) 1620 cm -1 (c) 1140 cm -1 and (d) 1580 cm -1 SERS band intensity calibrated in counts/mw/scan for 2-AMBT molecule at 1.0 x 10-5 M concentration.

108 99 However, for 3-days old colloid-molecule mixture at same adsorbate concentration, a significant decrease in intensity of the initial sol band is observed and the absorbance in the higher wavelength region gains further intensity and appears as a broad distinct band centered at around 665 nm. The appearance of this broad band in the longer wavelength region is attributed to the coagulation of colloidal silver particles in the presence of the adsorbed molecules [29-31]. The increase in absorbance in the longer wavelength region for the aged colloid-molecule mixture may be attributed to the proper aggregation of the mixture with time. Alternatively such a band has been ascribed to a charge transfer (CT) band due to molecule-metal interaction.[20,25,32] Substantial evidence regarding the ascription of CT band has been reported very recently by Fang et al [33]. The CT mechanism of SERS can be explained by the resonant Raman mechanism in which charge transfer excitations either from the metal to the adsorbed molecule or viceversa occur at the energy of incident laser frequency [34-35]. Molecule to metal CT excitation occurs when an electron is transferred from the highest occupied molecular orbital (HOMO) of the adsorbate to the Fermi level (E f ) of the metal. Conversely, transfer of an electron from the E F of the metal to the lowest unoccupied molecular orbital (LUMO) results in metal to molecule charge transfer. In order to introspect the direction of CT interaction, the HOMO energy of2-ambt has been estimated from the DFT calculation. The theoretical results shows that the HOMO energy of the molecule ~ ev which is energetically much lower than the E f of silver [36] (~ 5.48 ev). Hence, we conclude that metal to molecule CT interaction is more preferred in our case. Interestingly, the SERS spectra of the molecule at two different adsorbate concentrations in the different time domains are characterized by the enhancement of totally symmetric A / species. This may indicate that Albrecht A term i.e. the Frank- Condon term may play a dominant role in CT interaction [35, 37-39] Excitation Wavelength Dependence The excitation wavelength dependence of the SERS spectra of 2-AMBT at 1.0 x 10-5 M adsorbate concentration has been studied. The intensity of the well-resolved fairly intense bands at 860, 1140, 1580, 1620 cm -1 representing principally the inplane vibrations were investigated with four laser excitation wavelength (476.5, 488, 496.5, nm) available with our Ar + ion laser. The excitation power was kept constant for all wavelengths. The intense 921 cm -1 band of ACN was chosen as an

109 100 internal standard for intensity measurements. The results are shown in Figure-4.8 (II). It is observed that the surface enhancement for 2-AMBT molecule increases as the excitation wavelength shifts to longer wavelength. With our experimental limitation, we observe maximum enhancement on excitation with 514.5nm radiation from the argon ion laser, the longest wavelength available to us. This indicates that the resonance of the Raman excitation radiation with the new aggregation or CT band is more important than that with the original sol band at 392 nm for SERS activity. 4.3 Conclusion In the present chapter, the estimation of the vibrational frequencies of the free and different modeled surface complexes using DFT and ab initio (RHF) level of theory has been presented. These calculations allowed predicting the molecular structure of the surface complex, which closely resembles with actual SERS species. The orientations of the adsorbed species on colloidal silver surface have been estimated from surface selection rule. The estimated enhancement factors of the principal Raman bands indicates that in the surface adsorbed state, the 2-AMBT molecules are adsorbed on the silver surface through both the nitrogen atoms (N 9 and N 10 ) and is oriented with the molecular plane almost vertical to the surface.

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113 104 CHAPTER 5 Adsorption of 2-amino-6-methylbenzothiazole on colloidal silver particles: Quantum chemical calculations and surface enhanced Raman scattering study 5.1 Introductory Remarks Surface enhanced Raman Scattering (SERS) is emerging as a powerful spectroscopic tool for ultrasensitive chemical analysis down to single molecule level [1-2]. It has been widely used in studying the interaction mechanisms of molecules with the surface of substrate and molecular orientation [3-4]. The common analytical method is based on the shift of Raman bands, enhancement or weakening of intensity to hypothesize the adsorption orientation, geometry configuration and adsorption essence of adsorbates on the surface of the substrates. The studies on the experimental and theoretical NRS, SERS and FTIR of 2- Amino-6-methylbenzothiazole (2A-6MBT) molecule, are presented in this chapter. The spectral features of 2A-6MBT are different from 2ABT and 2A-4MBT presented in earlier chapters. The adsorptive behavior of 2A-6MBT molecules on the colloidal silver surface at different adsorbate concentrations has been elucidated from the SERS spectra. In these investigations, the silver surface may serve as an analogue for artificial biological interface, and after elucidating the adsorption mechanism of the molecule; the study can be extended to the adsorption on membranes or other interesting biological surface for medical or therapeutic treatments. DFT calculations on models of 2A-6MBT-Ag 0 and 2A-6MBT-Ag + surface complexes are also reported herein. 5.2 Results & discussion The preparation of silver sol, samples, the detail experimental technique and brief description of the used instruments are described in chapter 2 and 3.

114 Normal Raman and FTIR spectra of 2A-6MBT and their vibrational assignment Figure: 5.1 Optimized structure of 2-amino-6-methylbenzothiazole obtained at the BPW91/Lanl2DZ level of theory. The 2A-6MBT molecule has 19 atoms; hence, it has 51 fundamental vibrations. The molecule belongs to the C S point group. Simple group theory predicts that 35 planar (A / ) and 16 non-planar (A // ) species are expected to appear both in the Raman and in the IR spectra. The optimized structure of 2A-6MBT is shown in Figure 5.1. Selected optimized structural parameters of free and adsorbed 2A-6MBT molecules calculated by the DFT are listed in Table 5.1. From Table 5.1, it is clearly seen that the dihedral angles C 1 -C 2 -C 3 -N 9 and C 3 - N 9 -C 8 -N 10 are nearly ~ This indicates that the benzene ring and thiazole ring moieties of 2A-6MBT molecule are nearly planar. The nitrogen (N 10 ) and carbon (C 16 ) atoms of the exocyclic NH 2 and CH 3 groups of the molecule are sp 2 and sp 3 hybridized with the relevant bond angles ~ and ~ respectively.

115 106 Table 5.1 Relevant Structural Parameters of 2A-6MBT and the Change Therein upon Binding to Ag (Model IV) Calculated at BPW91/Lanl2DZ level of theory. DFT 2A-6MBT MODEL-IV Diff. Bond Lengths (A 0 ) C 2 -C C 3 -C C 4 -C C 6 -C C 3 -N C 8 -N C 8 -N C 8 -S C 4 -S C 5 -H Interatomic Angles (deg) C 3 -C 2 -C C 4 -C 3 -N C 3 -N 9 -C C 4 -S 7 -C C 6 -C 16 -C N 9 -C 8 -N H 12 -N 10 -H C 8 -N 10 -C N 9 -C 8 -S H 17 -C 16 -H H 15 -C 2 -C Dihedral Angles (deg) C 1 -C 2 -C 3 -N C 3 -N 9 -C 8 -N C 6 -C 5 -C 4 -S The normal Raman spectra of 2A-6MBT in neat solid and in 0.1 M ACN solution are shown in parts (a) and (b) of Figure 5.2, respectively. The calculated normal Raman spectrum (NRS) is shown in Figure 5.2 (c). It is to be emphasized that the calculated Raman spectrum represents the vibrational signatures of molecules in

116 107 their gas phase. Hence, the experimentally observed Raman spectrum of the solid and solution may differ significantly from the calculated spectrum. Raman activity (A 0 4 /a.m.u.) (c) Normalized Raman intensity (b) 242 * * * Raman intensity (a.u.) (a) Wavenumber/cm -1 Figure 5.2: Normal Raman spectra of 2A-6MBT (a) in the solid state and (b) in 0.1 M solution (the asterisks denote the solvent bands) for λ exc =514.5 nm. Part (c) shows the theoretical gas-phase Raman spectrum calculated using the DFT method Moreover, the observed disagreement between the theory and the experiment could also be a consequence of the anharmonicity and the general tendency of the quantum chemical methods to overestimate the force constants at the exact equilibrium geometry [5]. Nevertheless, after applying the respective scaling factors on the ab initio and DFT normal mode calculations, the theoretical results reproduce

117 108 the experimental data well and allow us to assign the vibrational modes. The experimentally observed FTIR spectrum of the powder sample in KBr pellet is shown in Figure 5.3. FTIR, NRS and SERS band frequencies of the molecule along with their tentative vibrational assignments and probable Raman polarizability tensor elements are summarized in Table 5.2. The calculated frequencies are also enlisted in the same table. The modes arising from the stretching and bending vibrations of the benzene, and thiazole moieties of 2-A6MBT molecule and the scissoring, rocking, twisting and wagging modes of the externally attached NH 2 and CH 3 groups are identified. In assigning the vibrational frequencies, literature concerned with the normal co-ordinate analysis and vibrational assignments of the related molecules has been consulted [6-12]. The visual inspection of the normal modes of the molecule animated from the output files of ab initio and DFT calculations using Gauss View 3.0 and Molekel 4.2 program are considered. Figure 5.3: FTIR spectrum of 2A-6MBT of neat powder in KBr pellet

118 109 Table 5.2: Observed and calculated Raman, IR, and SERS bands of 2A-6MBT in Varied Environments and their tentative assignments: FTIR (obsd) NRS Solid NRS (calcd) (obsd) RHF DFT NRS soln (obsd) SERS (10-4 M) Tentative assignment Probable tensor element. Symmetry species 1640h vvw 1616ms - NH 2 Scissors α yy A / 1620s w 1604ms ν(c=c)+nh 2 scissors α yy A / 1600h 1591w vw 1582 ν(c=n)+nh 2 scissors α yy A / 1587s 1526vs 1533s ms 1550ms ν(c=n)+ ν(c=c) α yy A / 1497vw s -CH 3 osc A / 1462vs 1474w ms 1451vs -CH 3 osc A / 1435sh β(c H) + ν (C-C) 1409sh 1413vw CH 3 osc 1376vvw 1368vw vvw β(c H) + ν (C-C) 1305sh β(c H)+ -NH 2 rocking 1280ms 1277vs s 1280vs β(c H)+ -NH 2 rocking +ν(c-s) α yy A / 1255ms 1250w ms 1257vs β(c H) α yy A / 1200vvw 1182ms vw 1212vw β(c H) 1129sh β(c H) 1103ms 1122w vw 1136 β(c H) α yy A / 1150ms A A A A A / / / / /

119 110 Table 5.2 (Continued) FTIR (obsd) NRS Solid NRS (calcd) (obsd) RHF DFT NRS soln (obsd) SERS (10-4 M) Tentative assignment Probable tensor element. Symmetry species 1075h 1072w vvw -CH 3 osc. A / β(c H); -NH 2 rocking; ν(c=n) 1034vw vvw 1054vw β(c H) 996vvw 997vw CH 3 osc. A / γ(c H) 936vvw γ(c H) 906vw 886vw kink 912sh Ring breathing; α(c N C) 858vvw γ(c H) 812vs w α(c N C); i.p. ring deformation of thiazole moiety; β(c H) 731vvw w o.o.p. ring deformation α yz, α xz A // 700w 671vw ms ms α (C C C) 655vw 656vw o.o.p. ring deformation thiazole moiety A // 630vw i.p. ring deformation A / 605ms vvw 622w γ(c H);-NH 2 twisting 566ms 565ms vvw ν (C-S); -NH 2 rocking 528vvw 523ms w -NH 2 wagging α yz, α xz A // A A A A A A A A A A / / // // / // / / // /

120 111 Table 5.2 (Continued) FTIR (obsd) NRS Solid NRS (calcd) (obsd) RHF DFT NRS soln (obsd) SERS (10-4 M) Tentative assignment Probable tensor element. Symmetry species 502ms 501vvw ip. ring deformation A / 468ms w γ (C H); γ (N-H) A 435w 430vw w 450 γ (C H); -NH 2 twisting 415s w 423w α (C C C) 409s C-CH 3 bending.(i.p.) A / 354w NH 2 twist; o.o.p. ring bending; ϕ (C-C-C-N) CH 3 osc.; -NH 2 rocking; δ (C-NH 2 ); δ (C-CH 3 ) 247ms vw -CH 3 ocs. A // CH 3 ocs.; -NH 2 ocs. A / o.o.p. ring bending. A // 212sh ν(ag N) o.o.p. ring deformation A // C-NH 2 ; C-CH 3 o.o.p. bending A // A A A A A // // / // / // α: in-plane angle bending; β: in plane bend; ν: stretch, γ: out of plane bend, δ: in plane bending, ϕ: out of plane dihedral bend, osc.: oscillation, vs : very strong, s : strong, ms: medium strong, w: weak, vw: very weak, vvw: very very weak, sh: shoulder, h: hump

121 Concentration-dependent SERS spectra of 2A-6MBT Parts (a-f) of Figure 5.4 show the normalized SERS spectra of the 2A-6MBT molecule at varied adsorbate concentrations in the range 1.0 x 10-2 M to 5 x 10-5 M. Careful examination of the concentration dependent SERS spectra, suggest that the concentration range between 1.0 x 10-2 M and 1.0 x 10-4 M is most sensitive not only for large signal counts but also for interesting variations in the relative intensities and band frequencies. Intensity reversal, distinct splitting and considerable red shift are observed for the pair of bands centered at around 676 and 692cm -1 whose NRS counterpart in solution is at 705cm -1. Normalized Intensity (c ) 21 2 * * * Normalized Intensity (f) * * * Normalized intensity Normalized Intensity (b) 21 1 (a ) * * 674 * * * * Normalized Intensity Normaliz ed Intensity (e) 212 (d ) * * * * * * W avenumber / cm Wavenumber/cm -1 Figure 5.4: Normalized SERS spectra of 2A-6MBT in silver hydrosol at concentrations (a) 1.0x10-2 M, (b) 5.0x10-3 M, (c) 1.0x10-3 M, (d) 5.0x10-4 M, (e) 1.0x10-4 M, and (f) 5.0x10-5 M for λ exc =514.5 nm (*denote the solvent band). The ph value of the SERS solution is ~7.5 The concomitance of both these bands in the SERS spectra suggest that there exist two types of adsorbed species namely A and B on the colloidal silver surface [13-15]. The relative populations of these adsorbed species vary with solution concentration. The 676 and 692 cm -1 bands are ascribed to the in-plane angle bending

122 113 modes of the benzene moiety of A and B species of 2A-6MBT molecule, respectively. However, considerable ~ 29 cm -1 and 13 cm -1 red shifts of 676 cm -1 and 692 cm -1 bands respectively compared to the corresponding NRS may connote the fact that the adsorption of A species involve strong interaction while the B species embrace relatively weak interaction of the benzene ring moiety of the molecule in the adsorption process [14]. Species A are dominant at concentrated solution, while species B predominantly exists at dilute adsorbate concentrations. At the intermediate adsorbate concentrations, both these species co-exist. Figure 5.5: Bar diagram indicating the FWHM of the Raman bands centered at ~ (a) 1582 cm -1 and (b) 1604 cm -1 in NRS and SERS spectra

123 114 Conspicuous increase in bandwidth is observed for the bands centered at around 1582 cm -1 and at 1604 cm -1 in the SERS spectra between 5.0 x 10-3 M and 1.0 x 10-4 M adsorbate concentrations. The bands at 1582/1604 cm -1 are assigned to coupled vibrations principally contributing from C=N/C=C stretch of the thiazole/benzene moieties of 2A-6MBT molecule and scissoring motion of the exocyclic NH 2 group. A precise evaluation of the full width at half-maximum (FWHM) values is very enigmatic for these bands because of the presence of nearby components. However, after deconvolution, the FWHM values in the NRS and SERS spectra at varied adsorbate concentrations are presented as bar diagrams in Figure 5.5(a) and 5(b). The substantial broadening of these bands may indicate surface-ring π orbital interaction in the adsorption of 2A-6MBT molecule on colloidal silver surface, which may result in considerable overlap of the molecular and metal orbital [14, 16]. Alternatively, the increase in bandwidth can also be due to the superposition of vibrational signatures contributing from both the adsorbed A and B species of 2A- 6MBT molecule [17]. Considering the entire concentration dependent profile, we presume that both of these possibilities are prevalent in the SERS spectra. The fused thiazole moiety of the molecule can also bind to the colloidal silver surface through the non-bonding lone pair electrons of either the nitrogen (N 9 ) or sulfur (S 7 ) atom or through both of them. We can treat this problem by enumerating the negative charge density on each of these probable active sites [13]. The higher the negative charges density on the atom, the higher the probability of it to act as an adsorptive site for the silver substrate. Theoretical results estimated from DFT calculations show that the partial charges on the N 9 and S 7 atoms determined by the natural population analysis (NPA) are and 0.337, respectively. The negative charge density is thus observed to be more appreciable on the nitrogen (N 9 ) atom than on sulfur (S 7 ) atom, thereby indicating active participation of nitrogen (N 9 ) atom in the adsorption process. The appearance of a shoulder at around 212 cm -1 in the concentration-dependent SERS spectral profile, ascribed to Ag-N stretching vibration [18], indicates that the fused thiazole moiety of the 2A-6MBT molecule is indeed adsorbed through the ring nitrogen (N 9 ) atom via σ-bond formation. The linear coordination (σ-bonding) through the nitrogen lone pair electrons of the thiazole moiety of the molecule may result in considerable blue shift of 1584 cm -1 bands compared to its NRS counterpart in solution. An interesting observation can be drawn

124 115 regarding the presence of 1282 cm -1 band in the entire concentration dependent SER spectral profile. This band has a noticeable contribution from C-S stretching internal coordinate, and it exhibits essentially the same frequencies and band shapes in the adsorbed and bulk-phase environment. This evidence further substantiates insignificant involvement of the sulfur (S 7 ) atom in the adsorption process. 529 (525) cm (1544) cm (1602 ) cm (1636) cm -1 Figure 5.6: Cartesian displacement and calculated (BPW91/Lanl2DZ) vibrational modes of 2A-6MBT. The numbers in parentheses refer to the experimental value of the assigned band. Apart from the benzene and the thiazole ring moiety of 2A-6MBT molecule, the lone pair electrons on the nitrogen (N 10 ) atom of the externally attached NH 2 group can also be the possible active site in the adsorption mechanism. The partial atomic charge on this nitrogen atom determined by NPA according to DFT calculation is In the concentration-dependent SERS spectra, we observe considerable enhancement and variation in the intensities of the bands centered around 522 and 1582, 1604, 1616 cm -1, assigned to the NH 2 wagging and scissoring modes respectively. However among these bands, the 1582 and 1604 cm -1 bands are known to be overlapped with the C=N and C=C stretch of the thiazole and benzene moiety of the molecule respectively. In fact, the 1582 cm -1 band is considerably blueshifted (~9 cm -1 ) in comparison to it corresponding NRS spectra in solution. Interestingly, 1616 cm -1 band, appeared at low adsorbate concentrations in the SER

125 116 spectra, is significantly red shifted in comparison to its NRS counterpart. The Cartesian displacements and normal modes of selected vibrations involving the NH 2 group and benzene, thiazole moieties of the molecule calculated from DFT calculations are shown in Figure 5.6. However, appreciable enhancement, intensity variation of the above-mentioned modes and the dramatic positional shift of 1582 and 1616 cm -1 bands may be due to the resultant interaction of the nitrogen (N 9 and N 10 ) atoms of the thiazole moiety and NH 2 group of 2A-6MBT molecule. The involvement of both the endocyclic and exocyclic nitrogen atoms in the adsorption process may result in the appearance of a broad shoulder in the SERS spectra at ~ 212 cm Orientation of the 2A-6MBT molecule on the colloidal silver surface The overall broadening of the SER bands make difficult to understand the exact orientation of the adsorbed A and B species on the colloidal silver surface. However in order to have a general idea regarding the orientation of the 2A-6MBT molecule at different adsorbate concentrations, we estimate the apparent enhancement factors (AEF) of some selected Raman bands using the relation-5.1 AEF = σ SERS [C NRS ] / σ NRS [C SERS ] (5.1) C and σ represent the concentration and the peak area of the Raman bands measured from baseline. They are shown in table 5.3. Table 5.3: Apparent Enhancement Factors of Some Selected Raman Bands of 2A-6MBT NRS soln (cm -1 ) Symme try species Apparent enhancement factor at varied concentrations* 1.0x10-2 M 5.0x10-3 M 1.0x10-3 M 5.0x10-4 M 1.0x10-4 M 5.0x10-5 M 1636 A / AB AB AB AB 5.14x x A / AB 1.01x x x x10 4 CNM 1573 A / CNM 6.15x x x x10 5 CNM 1544 A / 0.5x x x x x10 4 AB 1280 A / 1.98x x x x x x A / 1.32x x x x x x A / 1.01x10 2 AB 1.43x x x x10 AB 1.05x x x x x A // CNM 2.37x x x x x10 3 *CNM: could not measure; AB: absent

126 117 2A-6MBT molecule is considered to be lying in the xy plane, where x and y correspond to the long and short axis of the molecule and z is the perpendicular to the molecular plane. For different stance of tilted adsorption of the molecule on the colloidal silver surface, the vibrations of both the in-plane A / species spanning as xx (when long axis of the molecule is tilted to the surface) or yy (when the short axis of the molecule is tilted to the surface). As well as the out-of-plane A // species spanning as yz and xz are expected to undergo significant enhancement. It is clearly seen from Table 5.3, that we obtain a moderate 2-5 orders of magnitude enhancement of all bands principally representing the in-plane vibrations of A / species of 2A-6MBT molecule almost at all adsorbate concentrations. However, in the entire concentration dependent SER spectral profile, no significant enhancement contributing from the out-of-plane vibrations of the A // species of the molecule are recorded though considerable red shift is observed for the in-plane angle bending [α(c-c-c)] mode of the benzene ring moiety of the molecule. These results together with the appearance of Ag-N stretching vibration suggest that 2A-6MBT molecules are adsorbed onto the colloidal silver surface through the nitrogen (N 9 and N 10 ) atoms of the thiazole moiety and also possibly through the externally attached NH 2 group with the molecular plane tilted with respect to the silver surface at all adsorbate concentrations. The A and the B species that are prevalent at 1.0x10-2 M and 5.0x10-4 M adsorbate concentrations respectively are thought to be adsorbed in such a way that the y axis (the short axis) of the molecule is tilted or remain pendant on the colloidal silver surface. This type of preferred tilted orientation favors the involvement of benzene ring moiety of the molecule in the adsorption process. The relative variation in intensity and enhancement factor of the bands principally representing the in-plane vibrations of the benzene and the thiazole moieties of the molecule between 5.0x10-3 M and 1.0 x 10-4 M adsorbate concentrations may signify fluxional motion of the two adsorbed species (ca species A and B ) on the colloidal silver surface. Since an unequivocal surface selection rule is not available for SERS, so we cannot ascertain from the experimental results, the exact tilt angle of the y-axis (short axis) of the molecule on the silver surface for the A and B species. Interesting conclusion can be drawn regarding the orientation of the exocyclic NH 2 group of the molecule at different adsorbate concentrations. At 1.0 x 10-2 M to

127 x 10-4 M adsorbate concentrations, considerable enhancement of the NH 2 wagging mode at ~ 522 cm -1 and the absence of the almost pure NH 2 scissoring modes indicate that the N-H legs of the externally attached -NH 2 group are nearly parallel to the silver surface. However at more dilute adsorbate concentrations (ca 1.0 x 10-4 M and 5.0 x 10-5 M), the wagging and the scissoring modes of the NH 2 group of the molecule are enhanced significantly. This may signify that at dilute adsorbate concentrations the N-H legs of the NH 2 group are neither flat nor perpendicular to the surface but are tilted DFT Calculations on models of surface complexes The calculations of model surface complexes constituted by molecules bound to metal atoms or ions provide useful information about the interaction between the adsorbate and metal substrate [19-20]. The calculated frequencies of 2A-6MBT silver complex models involving one or two silver atoms or ions are shown in Table 5.4. Table 5.4: Calculated frequencies of four probable model Complexes along with the experimentally observed SERS bands at 1.0 x 10-4 M adsorbate concentration SERS of 1.0 x 10-4 M MODEL-I Energy ( ev) MODEL-II Energy ( ev) MODEL-III Energy ( ev) MODEL-IV Energy ( ev)

128 119 Table 5.4 (Continued) SERS of 1.0 x 10-4 M MODEL-I Energy ( ev) MODEL-II Energy ( ev) MODEL-III Energy ( ev) MODEL-IV Energy ( ev) The main aim of these types of calculations is to identify the species really adsorbed on the silver surface by the best agreement with the SERS bands. DFT

129 120 calculations on four different model complexes (Model I to IV) have been carried out. In the first model of the surface complex (Model-I) the nitrogen (N 9 ) atom of the thiazole moiety of the molecule interacts with a neutral silver atom (Ag 0 ). While in the second model (Model-II) two neutral silver atoms (Ag 0 Ag 0 ), of which one is bound to the N 9 atom and the other one bound to the nitrogen (N 10 ) atom of the externally attached amino group, are considered. Model-III and Model-IV represent similar type of binding arrangement as in Models I and II respectively, except neutral silver atom/atoms (Ag 0 and Ag 0 Ag 0 ) are replaced by silver ion/ions (Ag + and Ag + Ag + ). DFT results suggest that thermodynamically Model-IV is most stable, in the ground state with it being ~ ev and ~ ev more stable than Model-I and Model-III respectively. Model-II and IV are nearly of equal energy. However, one can see from Table 5.4, the theoretically predicted frequencies for Model-IV complex almost match the experimental results within tolerable limit. HOMO (-5.76 ev) LUMO (-0.34 ev) LUMO+1( ev) Figure: 5.7: Calculated HOMO, LUMO, and LUMO+1 of 2A-6MBT with BPW91/Lanl2DZ level of theory (Isocontour 0.02 a.u.)

130 121 This result may indicate that Ag + ions of the colloidal silver surface take active part in the adsorption process of 2A-6MBT molecule. This immediately allows us to investigate the changes in structural parameter, which 2A-6MBT molecules suffer because of Ag + interaction. They are listed in Table Direction of CT mechanism in the SERS of 2A-6MBT molecule The CT mechanism of SERS can be explained by the resonant Raman mechanism in which charge transfer excitations from the metal to the adsorbed molecule or vice versa occur at the energy of the incident laser frequency [21-22]. The frontier orbital theory plays a significant role in the understanding of the CT mechanism of SERS [23]. Two types of CT mechanism are predicted. One is molecule to metal and the other is metal to molecule. Molecule to metal CT excitations occurs when an electron is transferred from the highest occupied molecular orbital (HOMO) of the adsorbate to the Fermi level (E F ) of the metal. Conversely, transfer of an electron from the E F of the metal to the lowest unoccupied molecular orbital (LUMO) results in metal to molecule charge transfer [24-25]. The theoretical results show that the HOMO, LUMO, and LUMO+1 energies of the molecule are ~ ev, ~ ev and ~ ev respectively which are energetically much lower than E F of the silver (~ ev) [26]. Hence, it is concluded that metal to molecule CT interaction is more preferred in this case. The electron is probably transferred from metal to the LUMO of the molecule. The HOMO, LUMO, and LUMO +1 orbitals of the molecule are shown in the Figure 5.7. It is observed from Figure 5.7 that LUMO is mainly localized in the benzene, thiazole ring moiety of the 2A-6MBT molecule and in the externally attached NH 2 group. The transfer of electron from the E F of silver to the LUMO of the molecule may perturb the electron charge density in the benzene, the thiazole ring moiety and the NH 2 group of the molecule. This may result in the positional shift of certain SERS bands principally contributing from these moieties and from the exocyclic amino group. Interestingly the SER spectrum of the molecule at varied adsorbate concentrations is characterized by the enhancement of totally symmetric A / species. This may indicate that Albrecht A term i.e. the Frank-Condon term may play a dominant role in the CT interaction. [22, 27-28]

131 Conclusion The adsorption behavior of biologically significant 2-amino-6- methylbenzothiazole molecules on colloidal silver particles has been investigated by SERS aided by various quantum chemical calculations. The vibrational frequencies of different modeled surface complexes have been estimated using DFT level of theory. DFT results suggest that Ag+ ions of the colloidal silver surface take active part in the adsorption process of the molecule. The existence of two types of adsorbed species has been inferred from the concentration-dependent SERS spectral profile whose relative population varies with solution concentration. The orientations of the adsorbed species have been estimated from SERS spectral profile. The direction of charge transfer (CT) contribution to SERS has been inferred from the frontier orbital theory.

132 123 Reference: 1. S. Nie, S. R. Emory, Science 275, 1102, (1997) 2. H. Xu, E. J. Bjerneld, M. Kall, L. Borjesson, Phys. Rev. Lett. 83, 4357, (1999) 3. P. K. Chang, T. E. Furtak, Surafce enhanced Raman scattering, Plenum Press: New York J. Chowdhury, M. Ghosh, T. N. Misra, Spectrochim Acta A 56, 2107, (2000) 5. G. Rauhut, P. Pulay, J. Phys. Chem. 99, 3093, (1995) 6. G. Cardini, M. Muniz-Miranda, V. Schettino, J. Phys. Chem. B 108, 17007, (2004) 7. G. Varsanyi, Vibrational spectra of benzene derivatives; Academic Press: New York, London, (1969) 8. M. Moskovits, J. Chem. Phys. 77, 4408, (1982) 9. J. A. Creighton, Surf. Sci. 124, 209, (1983) 10. B. Pergolese, M. Muniz-Miranda, A. Bigotto, J. Phys. Chem. B 108, 5698, (2004) 11. L-R Wang, Y. Fang, Spectrochim Acta A 63, 614, (2000) 12. J. F. Arenas, J. Soto, I. Lopez-Tocon, D. J. Fernandez, J. C. Otero, J. I. Marcos, J. Chem. Phys. 116, 7207, (2002) 13. J. Sarkar, J. Chowdhury, M. Ghosh, R. De, G. B. Talapatra, J. Phys. Chem. B 109, 12861, (2005) 14. P. Goa, M. J. Weaver, J. Phys. Chem. 89, 5040, (1985) 15. M. Takahashi, Y. Sakai, M. Fujita, M. Ito, Surf. Sci. 176, 351, (1986) 16. S. H. Cho, H. S. Han, D. J. Jang, K. Kim, M. S. Kim, J. Phys. Chem. 99, 10594, (1995) 17. M. Takahashi, M. Ito, Chem. Phys. Lett. 103, 512, (1984) 18. G. Cardini, M. Muniz-Miranda, V. Schettino, J. Phys. Chem. B 108, 17007, (2004)

133 B. Pergolese, M. Muniz-Miranda, A. Bigotto, J. Phys. Chem. B 108, 5698, (2004) 20. L-R Wang, Y. Fang, Spectrochim. Acta. A 63, 614, (2000) 21. A. Campion, P. Kambhampati, Chem. Soc. Rev. 27, 241, (1998) 22. J. F. Arenas, J. Soto, I. Lopez-Tocon, D. J. Fernandez, J. C. Otero, J. I. Marcos, J. Chem. Phys. 116, 7207, (2002) 23. R. L. Garell, J. E. Chadwick, D. L. Severance, N. A. Mc Donald, D. C. Myles, J. Am. Chem. Soc. 117, 11563, (1995) 24. A. R. Bizzarri, S. Cannistraro, Chem. Phys. 290, 297, (2003) 25. A. R. Bizzarri, S. Cannistraro, Chem. Phys. 395, 222, (2004) 26. C. Kittel, Introduction to solid state physics, 5th ed.; Wiley: New York, p 154 (1976) 27. J. R. Lombardi, R. L. Birke, T. Lu, J. Xu, J. Chem. Phys. 84, 4174, (1986) 28. R. L. Birke, T. Lu, J. R. Lombardi, Technique for Characterization of electrodes and electrochemical process; John Wiley & Sons.; Chapter 5, (1991)

134 125

135 125 CHAPTER 6 Ab initio, DFT vibrational calculations and SERRS study of Rhodamine 123 adsorbed on colloidal silver particles 6.1 Introductory Remarks Application of quantum chemical calculations in the adsorption behavior of molecules on metal surface and assignment of vibrational modes in Raman spectroscopy is very interesting field. Detailed ab initio and DFT analysis of Rhodamine 123 molecule is rare. Rhodamine 123 (Rh123) is a biologically important molecule. It has been extensively employed as a fluorescence stain of mitochondria in living cell [1-2]. Its halogenated analogues are used as phosensitizer in Photodynamic therapy [3]. In this chapter, we investigate the adsorptive behavior of Rh123 on the colloidal silver surface and the nature of charge transfer between the molecule and the metal using FTIR and SERRS spectra together with ab initio and DFT calculations. This study may be helpful to understand the role of this molecule at biological interfaces. 6.2 Results & Discussion The preparation of silver sol, samples, the detail experimental technique and brief description of the used instruments are described in chapter 2 and Molecular Geometry of Rh123 The molecular structure of cationic Rh123 was optimized by ab initio RHF and DFT theories. Selected optimized structural parameters of Rh123 molecule are listed in table-6.1. In the process of geometry optimization for the fully relaxed method, convergence of all the calculations and the absence of imaginary values in the wave numbers confirmed the attainment of local minima on the potential energy surface. It is observed from table-6.1 the dihedral angles C 6 -C 5 -O 10 -C 9 and O 10 -C 9 - C 14 -C 13 are nearly~ These indicate that the xanthene ring moiety of Rh123 molecule is almost planar in its electronic ground state.

136 126 Table 6.1: Relevant Structural Parameters of Rh123 molecule calculated from RHF/6-31G and B3LYP/6-31G levels of theories. RHF DFT Bond lengths (Å) C 1 C C 5 C C 2 H C 1 N C 4 C C 5 O N 26 C N 26 H C 7 C C 15 C C 20 C C 21 C C 21 O O 23 C Bond angles (degree) C 6 C 1 C C 6 C 1 N C 6 C 5 O C 1 C 2 H C 4 C 7 C C 5 O 10 C H 43 N 26 H C 15 C 20 C C 15 C 20 C C 21 O 23 C O 23 C 21 O Dihedral angles (degree) C 6 C 1 N 25 H C 4 C 7 C 15 C C 7 C 8 C 11 C C 19 C 20 C 21 O The estimated dihedral angles C 6 -C 1 -N 25 -H 40 and C 14 -C 13 -N 26 -H 42 are also ~ These indicate that the externally attached amino groups lie in the plane of the xanthene ring moiety of the molecule. Geometry optimizations, using the abovementioned level of theories further suggest that the angle between the xanthene plane and its adjacent phenyl-ring plane is nearly perpendicular (~-92.4/ in the RHF/DFT), whereas the angle in the crystal structure is ~ [1]. This is in

137 127 accordance with the recently reported structural parameter of Rhodamine 6G (R6G) molecule [4]. The DFT optimized structure of Rh123 molecule is shown in Figure Normal Raman, FTIR spectra and vibrational analysis of Rh123 Figure 6.1: The optimized structure of Rhodamine123 molecule obtained from B3LYP/6-31G level of theory Rhodamine 123 molecule has 43 atoms; hence it has 123 fundamental vibrations. It belongs to C S point group and consequently, 83 planer (A / species) and 40 nonplaner (A // species) fundamental vibrations are expected to appear both in the Raman and in the FTIR spectra. The normal Raman spectra of Rh123 molecule at 3.0x10-4 M aqueous solution is shown in Figure 6.3 (c). Figures 6.3 (a) and (b) represent the theoretical NRS spectrum of Rh123 molecule calculated from ab initio (RHF) and DFT levels of theories. It is to be emphasized that the calculated Raman spectrum represents vibrational signatures of molecules in their gas phase.

138 128 Hence, the experimentally observed Raman spectrum of the solid and solution may differ significantly from the calculated spectrum [5]. Moreover, the ab initio (RHF) calculations typically predict larger harmonic vibrational wave numbers than the ones observed experimentally [6]. Thus, the restricted Hartree-Fock (RHF) vibrational wave numbers presented in Table-2 have been uniformly scaled by the scaling factor of [7]. In DFT calculations, the B3LYP functional also tends to overestimate the fundamental modes compared to the experimentally observed values [7, 8]. In order to obtain a considerable better agreement with the experimental data, scaling factors have to be used [7, 8]. Thus, a scaling factor of has been uniformly applied to the B3LYP calculated wave numbers [7]. The observed disagreement between the theory and the experiment could be a consequence of the anharmonicity and of the general tendency of the quantum chemical methods to overestimate the force constants at the exact equilibrium geometry [9] Absorbance Wavenumber/cm -1 Figure 6.2: FTIR spectrum of Rh123 molecule of neat powder in KBr pellet. Nevertheless, after applying the respective scaling factors on the ab initio and DFT normal mode calculations, as one can see from Table-6.2, the theoretical calculations reproduce the experimental data well and allow us to assign the vibrational modes. The observed FTIR spectrum of the powder sample in a KBr pellet

139 129 is shown in figure-6.2. Table 6.2 lists the FTIR, NRS and SERS band frequencies of the molecule along with their tentative assignments and symmetry species. Table 6.2: Observed and Calculated Raman, IR and SERS bands of Rh123 molecule and their tentative assignments* FTIR (obs) NRS solu (obs) NRS (calc) RHF DFT SERS Tentative assignment Symmetry species m XR Stretching A / w XR Stretching A / 1578sh PHR Stretching A / 1558m sh XR Stretching A / w XR Stretching A / 1500s XR Stretching A / XR Stretching A / XR Stretching A / s XR Stretching A / m C-H bending of XR A / C-H bending of XR A / w C-H bending of XR A / m C-H bending of XR A / PHR Stretching A / C-H bending of XR A / 940w XR +PHR Stretching A / w XR +PHR Stretching A / C-H o.p. bending of XR A // C-H o.p. bending of XR A // m C-H o.p. bending of XR A // C-H o.p. bending of PR A // C-H o.p. bending of XR A // m XR +PHR Stretching A / NH 2 wagging A // C-H o.p. bending of XR A // 418s XR deformation + NH 2 A / Oscillation. 343s XR Stretching A / *XR: xanthene ring; PHR: phenyl ring; o.p.: out of plane. In assigning the vibrational frequencies of Rh123 molecule, the visual inspection of the normal modes animated from the output files of ab initio and DFT calculations using Gauss View 3.0 and Molekel 4.2 program have been considered.

140 130 The available literature concerning the vibrational assignment of this [10] and related molecules [4, 11] are also consulted.. In tensity (a.u.) (d) (c) Intensity (a.u.) Raman activity(a 0 4 /a.m.u.) (b) Raman acti vity (A 0 4 / a.m.u.) (a) Wavenumber/cm -1 Figure 6.3: Background-corrected (a) the theoretical gas-phase Raman spectrum calculated using RHF method; (b) using DFT method; (c) Raman spectra of aqueous solution (3.0x10-4 M) of Rh123 at ph 6.5 for λ exc = 609 nm; (d) SERS spectrum of 3.0x10-7 M Rh123 adsorbed in silver hydrosol at ph 2 for λ exc = nm.

141 131 The modes principally arising from the xanthene, phenyl rings and the externally attached NH 2 group of Rh123 molecule are identified. Interesting observation can be drawn regarding the assignments of some vibrational bands centered at around 1500 (calculated 1498/1496 cm -1 in DFT/RHF) and 1592 cm -1 (calculated 1606/1596 cm -1 in DFT/RHF). The 1592 cm -1 mode is strong in the FTIR but appears as weak but prominent band in the observed NRS. The other band at 1500 cm -1 is strong in the experimental NRS but does not appear in the FTIR spectra. The visual inspection of these normal modes indicates that the vibrations principally represent the stretching vibrations of the xanthene ring moiety of the molecule. Previously these bands were ascribed to externally attached phenyl ring stretching vibration of Rh123 molecule. There is a discrepancy in the assignment of another band centered at 1272 cm -1 (calculated 1278/1290 cm -1 in DFT/RHF). 602 (597 cm -1 ) 1278 (1273cm -1 ) 1498 (1507cm -1 ) 1606 (1601cm -1 ) Figure 6.4: Cartesian displacement and calculated (B3LYP/6-31G) vibrational modes of Rh123. The numbers in the parentheses referred to the experimental value of the assigned band

142 132 This band is prominent both in the Raman and in the FTIR spectra and has been assigned to the in-plane C-H bend of the xanthene ring moiety of the molecule. Previously, this band was assigned to the C-O-C stretching mode of the xanthene ring of rh123 molecule [10]. Inference can be drawn regarding the assignments of the vibrational bands centered at around 662 cm -1 (calculated 659/665 cm -1 in DFT/RHF), 705 cm -1 (calculated 707/703 cm -1 in DFT/RHF), 812 cm -1 (calculated 820/804 cm -1 in DFT/RHF) and 848 cm -1 (calculated 850/855 cm -1 in DFT/RHF). All these bands are absent in the Raman spectrum but appear in FTIR. Generally, out-of-plane modes appear strong in the infrared and weak in the Raman [12-13]. Considering this fact, 662, 812, 848 cm -1 and 705 cm -1 bands are ascribed to C-H out-of-plane bend of the xanthene ring and the phenyl ring respectively of Rh123 molecule. The visual inspection of the above modes also substantiates this conjecture. The Cartesian displacements and normal modes of some selected vibrations calculated from DFT calculation are shown in figure SERRS spectra of Rh123 The SERRS spectra of Rh123 molecule at 3.0x10-7 M adsorbate concentration at ph 2 with nm excitation is shown in figure 6.3(d). The SERRS spectra oh Rh123, recorded under identical experimental condition at neutral ph, is characterized by strong fluorescence background overshadowing the Raman signal. The fluorescence background is reduced significantly at ph 2 with the appearance of sharp Raman bands. Detailed analyses of the ph dependent SERRS spectra of Rh123 molecule adsorbed on silver hydrosol are reported elsewhere [10]. Scanning through the SERS spectra, immediately reveal that the prominent SERS bands principally represent the in-plane vibrational modes of the xanthene and the phenyl rings moieties of Rh123 molecule. Significant enhancements are observed for the bands centered at around 1650, 636 and 352 cm -1 in the SERRS spectra assigned to the stretching vibrations of the xanthene ring moiety of the molecule. In fact, these bands also show 8 to 10 cm -1 blue shift in comparison with its NRS counterpart in solution. Interestingly we observe that the vibrational signatures principally contributing from extremal phenyl ring moiety of the molecule in the SERES spectra exhibit the same band shapes and show very small or no shift compared to its NRS counterpart in solution. These observations may signify considerable interaction of the xanthene ring moiety and weaker or no interaction of

143 133 extremal phenyl ring moiety of Rh123 molecule with the colloidal silver surface. The xanthene ring moiety of the molecule, however, can bind to the silver surface through the lone pair electrons of oxygen (O 10 ) and nitrogen (N 25 and N 26 ) atoms of the externally attached amino group. In order to find the probable adsorptive sites of the molecule on the silver surface, we estimate the negative charge density on each of these atoms [14-15]. The higher is the negative charge density on the atom, the higher is the probability of it to act as an adsorptive site for silver substrate. Theoretical results estimated from DFT/RHF ab initio calculations show that the Mullikan atomic charges on the N 25, N 26, and O 10 atoms are 0.788/-0.980, /-0.980, and 0.577/ respectively. Thus, the negative charge density is observed to be appreciable on both the nitrogen atoms as well as on the oxygen atom of the xanthene ring moiety of the molecule. The theoretical results, therefore predict active involvement of N 25, N 26, and O 10 atoms of Rh123 molecule in the adsorption process, though the existence of Ag-N or Ag-O stretching vibrations are not recorded in the SERS spectra Electronic absorption spectra of silver colloid with added Rh123 Figure 6.5: Room temperature UV-vis absorption spectra of (a) pure silver sol; (b) sol with added Rh123 (concentration 3.0 x 10-7 M) at ph 2

144 134 Figure-6.5 shows the room temperature UV-visible electronic absorption spectra of Ag sol, as prepared, before and after the addition of Rh123 molecule at 3.0 x 10-7 M adsorbate concentration. The pure stable silver sol shows a single extinction maximum at 394 nm. When 3.0 x 10-7 M Rh123 is added to the sol at ph2, the extinction maximum at 394 nm diminishes with the appearance of a broad hump in the longer wavelength region. The appearance of this broad hump at longer wavelength region is attributed to the coagulation of colloidal silver particles in the presence of the adsorbed molecules [16-17]. Alternatively, such a band has been ascribed to a charge transfer (CT) band due to molecule-metal interaction [18-20]. The frontier orbital theory plays a significant role in order to understand the CT mechanism of SERS and SERRS [21-22]. Two types of CT mechanisms are predicted. One is molecule to metal and other is metal to molecule. Molecule to metal CT excitation occurs when an electron is transferred from highest occupied molecular orbital (HOMO) of the adsorbate to the Fermi level (E F ) of the metal. Conversely, transfer of an electron from E F of the metal to the lowest unoccupied molecular orbital (LUMO) results in metal to molecule charge transfer. In order to introspection the direction of CT interaction, the HOMO, LUMO, LUMO+1 energies of Rh123 molecule have been estimated from the DFT calculation. The theoretical results show that the HOMO, LUMO, and LUMO+1 energies of the molecule are ~ ev, ~ ev and ev respectively which are energetically much lower than the E F of the silver (~ ev) [23]. Hence, we conclude that metal to molecule CT interaction is more preferred in our case. The electron is probably transferred from metal to the LUMO of the molecule. The HOMO, LUMO and LUMO+1 orbital of the molecule are shown in figure-6.6. It is observed that the HOMO and LUMO are mainly localized in the xanthene ring moiety and LUMO+1 is localized on the phenyl ring moiety of Rh123 molecule. The transfer of electron from the Fermi level of silver to the LUMO of the molecule may perturb the electronic charge density in the xanthene ring of the molecule. This may results in the blue shift of certain SERRS bands principally contributing from the xanthene ring moiety of Rh123 molecule. Interestingly, the SERR spectrum of the molecule is characterized by the enhancement of totally

145 135 symmetric A / species. This may indicate that Albrecht A term i.e. the Frank-Condon term may play a dominant role in CT interaction [24-25]. φ 92 (LUMO+1) φ 91 (LUMO) φ 90 (HOMO) Figure 6.6: Calculated (i) HOMO (ii) LUMO (iii) LUMO + 1 of Rh123 with B3LYP/6-31 G (Isocontour 0.02 a.u.). 6.3 Conclusion The adsorption behavior on colloidal silver particles of enormous biologically importance Rhodamine 123 molecules has been investigated by SERRS spectroscopy.

146 136 The observed Raman and infrared bands of this molecule are satisfactorily assigned based on Ab initio (RHF) and DFT calculations employing a high-level basis set. Some vibrational modes of the free molecule have been reassigned based on above mentioned visualization program. From HOMO, LUMO energies calculations, we conclude that metal to molecule CT interaction is more preferred in this case and enhancement calculations predicts that in the CT interaction, Albrecht A term play a significant role.

147 137 Reference: 1. I. C. Summerhayes, T. J. Lampidis, S. D. Bernal, J. J. Nadakavukaren, K. K. Nadakavukaren, E. L. Shepard and L. B. Chen, Proc. Natl. Acad. Sci. USA 79, 5292, (1982) 2. L. Villeneuve, P. Pal, G. Durocher, D. Girard, R. Giasson, L. Blanchard an L. Gaboury, J. Fluorescence 6, 209, (1996) 3. B. W. Henderson and T. J, Douglherty, Photochem, Photobiol. 55, 145, (1992) 4. H. Watanabe, N. Hayazawa, Y. Inouye, S. Kawata, J. Phys.Chem.B 109, 5012, (2005) 5. R. F. Aroca, R. E. Clavijo, M.D. Halls, H. B. Schlegel, J. Phys. Chem.A 104, 9500, (2000) 6. W. J. Hehre, L. Random, P. V. R. Schleyer, J. A. Pople, In Ab Initio Molecular Orbital Theory, J. Wiley, New York, (1986) 7. A. P. Scott, L. Radom, J. Phys. Chem. 100, 16502, (1996) 8. M. W. Wang, Chem. Phys. Lett. 256, 391, (1996) 9. G. Rauhut, P. Pulay, J. Phys. Chem. 99, 3093, (1995) 10. J. Chowdhury, P. Pal, M. Ghosh, T. N. Misra, J.Colloid and Interface Sci.235, 317, (2001) 11. P. Hildebrandt, M. Stockburger, J. Phys. Chem. 88, 5935, (1984) 12. S. C. Wait, J. C. Menerney, J. Mol. Spectrosc. 34, 56, (1970) 13. J. Sarkar, J. Chowdhury, M. Ghosh, R. De, G. B. Talapatra, J. Phys. Chem. B 109, 12861, (2005) 14. J. Chowdhury, K. M. Mukherjee, T.N. Misra, J.Raman Spectrosc. 31, 427, (2000) 15. M. Baia, L. Baia, W. Kiefer, J. Popp, J. Phys. Chem. B 108, 17491, (2004) 16. J. A. Creighton, C. G. Blatchford, M. G. Albrecht, J. Chem. Soc. Faraday Trans. 275, 790, (1979) 17. J. V. Garcia-Ramos, S. Sanchez-Cortes, J. Mol. Struc. 405, 13, (1997)

148 J. Chowdhury, M. Ghosh, T. N. Misra, Spectochim. Acta, A 56, 2107, (2000) 19. S. Sanchez-Cortes, J. V. Garcia-Ramos, G. Morcillo, A. Tinti, J. Colloid Interface Sci. 175, 358, (1995) 20. Y. Du, Y. Fang, Spectrochim. Acta, A.60, 535, (2004) 21. R. L. Gavell, J. E. Chadwick, D. L. Severance, N. A. McDonald, D. C. Myles, J. Am. Chem. Soc. 117, 11563, (1995) 22. A. Campion, P. Kambhampati, Chem. Soc. Rev. 27, 241, (1998) 23. C. Kittel, Introduction to solid state physics, Wiley Eastern Publication, 5 th edition, page J. R. Lombardi, R. L. Birke, T. Lu, J. Xu, J. Chem. Phys. 84, 4174, (1986) 25. J. A. Creighton, Surf. Sci. 173, 665, (1986)

149 139 CHAPTER 7 Adsorption of 4-Methyl-4H-1, 2, 4-triazole-3-thiol Molecules on Silver Nanocolloids: FTIR, Raman, and Surface-Enhanced Raman Scattering Study Aided by Density Functional Theory 7.1 Introductory Remarks Normal Raman scattering, however is a weak process, characterized by cross sections of ~ cm 2. Therefore, normal Raman scattering is often interfered with fluorescence emission. The potential to combine sensitivity of fluorescence with the structural information content makes Surface enhanced Raman scattering (SERS) spectroscopy a powerful tool in a variety of fields including bio spectroscopy [1-2]. It has become an increasing popular technique not only for studying the molecules or ions at trace concentrations down to single molecule detection level [3-4], but also in estimating the molecular forms and their possible orientation on the metal surface [5-7]. Considering the enormous industrial and biological importance of Azoles and its derivatives, we present in this chapter the detailed experimental and theoretical normal Raman spectra (NRS), SERS and FTIR spectra of 4-Methyl-4H-1, 2, 4- Triazole-3-Thiol (4-MTTL) molecule. From a more fundamental point of view, 4- MTTL is also very interesting compound because of its probable existance in thionethiol tautomeric equilibrium in electronic ground state. The ph dependent NRS spectra of the molecule in aqueous solution have been recorded to elucidate the protonation effect and preferential exitence of the tautomeric form/forms of the molecule in acid, neutral and alkaline media. The adsorptive behavior and the orientation of the molecule on the nanocolloidal silver surface at various ph values are also recorded herein. 7.2 Results & Discussion Molecular structure 4-MTTL molecules can exist in thione and in anionic forms. The optimized molecular structures of the molecule in three different forms are shown in Figure 6.1. In order to retrieve some ideas concerning about the relative stability of different forms of the molecule in gas phase, minimum energies of the molecule at their respective optimized geometries have been computed using DFT method. The theoretical results indicate that both the thiol and the thione forms of the molecule are

150 140 energetically more favorable than its anionic form. The corresponding energies at global minima of the potential energy surfaces of different forms of the molecule are also shown in Figure 7.1. Table 7.1 shows the selected optimized structural parameters, and rotational constants of three different molecular forms. (a) Energy ( ev) (b) Energy ( eV) (c) Energy ( eV) Figure 7.1: The optimized molecular structure of the (a) Thione (b) Thiol and (c) Ionic forms of the molecule obtained from B3LYP /6-31G (d, p) level of theory Table 7.1: Relevant Structural Parameters and rotational constants of thiol, thione, and anionic forms of the molecule calculated from B 3LYP /6-31G (d, p) level of theory. thiol thione ionic Bond Length (Å) N 1 -N N 1 -N N 1 -N N 2 -C N 2 -C N 2 -C C 3 S C 3 S C 3 S N 4 -C N 2 -H N 4 -C Bond Angle (deg) N 1 -N 2 -C N 1 -N 2 -C N 1 -N 2 -C N 2 - C 3 -S N 2 - C 3 -S N 2 - C 3 -S C 3 -S 6 -H N 1 -N 2 -H S 11 -C 3 -N H 10 -C 7 -H H 9 -C 7 -H H 8 -C 6 -H N 2 -C 3 -N N 2 -C 3 -N N 2 -C 3 -N Dihedral Angle (deg) N 1 -N 2 -C 3 -S N 1 -N 2 -C 3 -S N 1 -N 2 -C 3 -S N 2 -C 3 -S 6 -H S 6 -C 3 -N 2 -H C 3 -N 4 -C 5 -H C 5 -N 1 -N 2 -C C 5 -N 1 -N 2 -C C 5 -N 1 -N 2 -C S 6 -C 3 -N 4 -C S 6 -C 3 -N 4 -C S 11 -C 3 -N 4 -C Rotational Constant (GHz) thiol thione ionic A B C A B C A B C

151 141 To the best of our knowledge, no electron diffraction or microwave data of this molecule has yet been established. However, the theoretical results estimated from the DFT calculations are almost comparable with the reported X-ray crystallographic data of the molecule [8]. The triazole moieties of all three probable forms of the molecule are planar, which is consistent with the literatures published elsewhere [9]. The carbon atom of the exocyclic CH 3 group of the thione, thiol and anionic forms of the molecule is sp 3 hybridized with the relevant bond angle ~ , and degree respectively. The Hydrogen atom (H 9 ) of 4-MTTL molecule lies above the plane of the triazole moiety of the molecule with C 3 S 6 H 9 bond angle is ~ 97.19º and N 2 C 3 S 6 H 9 dihedral angle is ~ 94.89º Normal Raman and FTIR Spectra of the molecule and their Vibrational Assignment 4-Methyl-4H-1, 2, 4-Triazole-3-Thiol (4-MTTL) molecule and its thione (4- MTTN) form have 12 atoms; hence, both of them have 30 fundamental vibrations. The tautomeric forms of the molecule belong to C s point group symmetry. So, 21 planar (A / species) and 9 non-planar (A // species) fundamental vibrations are expected to appear in the Raman and in the FTIR spectra of both the thione and the thiol forms of the molecule. However, among these vibrations originating from the tautomeric forms of the molecule, some vibrations are degenerate. The FTIR spectrum of powdered molecule in KBr pellet is shown in Figure 7.2a. The corresponding NRS of the neat solid is depicted in Figure 7.2b. The calculated NRS spectra of the thiol and the thione forms of the molecule in gas phase are shown in the panels, c and d of Figure 7.2 respectively. The underlying aim of recording the NRS and FTIR spectra is to apprehend the existence of preferential tautomeric form/forms of the molecule from the assignment of the vibrational signatures. Table-7.2 lists the experimentally observed FTIR and NRS band frequencies of the molecule. The theoretically computed vibrational frequencies of the thiol and the thione forms of the molecule in gas phase are also shown in Table-7.2 along with their tentative assignments, as provided from the potential energy distributions (PED). The PED calculations in terms of internal coordinates of the molecule have been estimated from the output of the DFT calculations. The observed disagreement between the theory and the experiment could be a consequence of the anharmonicity and may be due to the general tendency of the quantum chemical methods to overestimate the force constants at the exact equilibrium geometry [6-7]. However, it is to be emphasized that the calculated Raman spectrum represents the vibrational signatures of molecules in its gas phase. Hence, the experimentally observed NRS of solid and solution may differ significantly from the calculated spectrum. Despite of the fact, one can see that there is a general

152 142 concordance regarding the Raman intensities as well as the position of the peaks between the experimental and calculated spectra [10-11]. (d) Raman activity (A 0 4 /a.m.u.) (c) Raman activity (A 0 4 / a.m.u.) (b) Raman intensity (a.u.) Absorbance Wavenumber / cm (a) Wavenumber / cm -1 Figure 7.2: a) FTIR spectrum of the molecule of neat powder in KBr pellet b) Normal Raman spectra of the molecule in solid state (λ exc = nm). The theoretical gas-phase Raman spectrum of the thiol (c) and thione (d) forms of the molecule calculated using B3LYP /6-31G (d, p) level of theory

153 143 Table 7.2: Observed and calculated IR and Raman bands of the molecule in varied environment and their tentative assignments FTIR (obs) NRS NRS in Solution at different ph Calculated Assignment (PED) b Calculated Assignment Solid ph-2 ph-4 ph-7 ph-9 freq. of 4- freq. of 4- (PED) b MTTL MTTN 253s 350s 409w α 3,4,7 34α 9,6,3 8r 3, vw 423w 414 w 415w 412vw α 12,2,3 511sh 511 vvw 512sh r 3,6 17γ 6,9,4,3 14α 9,6, ms 535sh 525ms 531w γ 12,2,3,6 10τ 5,1,2,3 548ms 540 sh 540sh 540ms 540w r 3,6 14 α 10,4,7 671ms 671w 664 ms 661sh 665vvw τ 12,2,1,5 696sh 699vs 697 s 697s 695vs 697s r 4,7 22α 3,4,5 9r 3, r 4,7 33α 12,2,3 6α 8,1,5 852w 836 ms 836ms γ 1,8,2,5 9τ 5,1,2,3 6τ 3,4,5, γ 1,8,2,5 949s 943ms 957 ms 951s 952s γ 6,9,4,3 39α 3,9,6 7α 4,6, α 12,2,3 27α 2,1,5 964w 964w α 5,2,1 11r 3,4 10α 1,2,3 1042s 1045vw 1059w 1066w 1052vw r 1, α 10,4,7 23α 10,11,7 1156ms 1154ms 1165w 1168ms 1164w α 11,4,7 20α 10,4, α 1,3,2 18r 3, α 10,11,7 30α 10,7,4 53α 12,2,3 33α 9,7,4

154 144 Table 7.2 (Continued) FTIR NRS NRS in Solution at different ph Calculated Assignment (PED) b Calculated Assignment (obs) Solid ph-2 ph-4 ph-7 ph-9 freq. of 4- MTTL freq. of 4- MTTN (PED) b ph s 1217s 1204, 1236w 1227w α 8,1,5 11r 1,5 8α 1,3, w 1276s 1261ms 1266w 1262w (α 8,5,1 ; α 2,1,5 ) 1294s 1297ms 1294ms 1290w 1326s 1334s 1320w 1317vvw 1345ms 1345ms 1342s 1349ms α 10,11,7 24α 12,2,3 1361ms 1365ms r 3,4 15r 4,5 12α 5,2, ms 1373ms 1373ms 1370w 1386ms r 2,3 20r 4,7 18r 1,5 18r 4,5 1393ms 1395ms 1399w 1397sh α 11,4,7 30α 8,5,1 1427sh 1436w 1436ms 1436ms 1437ms 1450ms α 10,11,7 28r 2,3 7r 4, w α 10,11,7 27r 2,3 18r 1, α 10,11,7 17α 11,7,4 1489vs 1495ms 1508ms 1507s 1509vs α 10,12,7 37α 11,12,7 8α 12,4, α 10,11, vs 1554ms 1541vvw 1542vw r 1,5 20α 8,1,5 15r 4,5 9r 3, ( 85α 10,11,7 7α 12,2,3 ) 1563ms 1571w

155 145 Table 7.2 (Continued) FTIR NRS NRS in Solution at different ph Calculated Assignment (PED) b Calculated Assignment (obs) Solid ph-2 ph-4 ph-7 ph-9 freq. of 4- MTTL freq. of 4- MTTN (PED) b 1609ms 1609ms r 1,5 1635ms 1627vw 1664s r 6,9 26γ 6,9,4,3 / N-H.H str r 7,11 23r 7,10 17r 7, r 7,10 30r 7, r 7,10 39r 7,11 15r 7, r 7,12 31r 7, r 7,9 26r 7, r 5, r 5, r 2,12 vs:very strong;s:strong;ms:medium strong;w: weak; vw:very weak; vvw:very very weak; sh:shoulder; b r: stretching; α: in-plane bending; γ: out-of-plane bending; τ: torsion; only contributions 5 are reported.

156 NRS and FTIR spectrum of the molecule in solid state The FTIR of the powdered sample and the NRS spectra of the solid state are characterized by sharp and well-resolved vibrational bands (panels a, and b of Figure 7.2). The modes arising principally from the stretching and bending vibrations of the 1, 2, 4 triazole moiety of both the thiol and the thione forms of the molecule and the externally attached-ch3 group are identified. In assigning the vibrational frequencies, literatures concerning with the normal coordinate analysis and vibrational assignments of the related molecules have been considered [9, 12-15]. Interesting observations can be drawn regarding the appearance of bands at around 535 and 548 cm -1, whose IR counterparts show a weak but well resolved broad absorption band at ~ 538 cm -1. Both these modes have significant contribution from the stretching, torsions and bending vibrations of the thione form of the molecule. The other rings bending vibrational modes emanating from the thione form of the molecule are observed at 1276 and 1395 cm -1 in the NRS spectra in the solid state whose IR counterparts are recorded at 1268 and at 1393 cm -1 respectively. The appearance of the vibrational signatures contributing from the torsion, bending and stretching vibrations of the 1, 2, 4 triazole moiety of the thione form of the molecule in the NRS and in the IR spectra allow us to predict that the thione form of the molecule exists in the solid state. However, the Raman and the IR spectra in the solid state are also characterized by the presence of bands centered at around 964 cm -1 ascribed to in-plane ring bending vibration of the triazole moiety of the thiol form of the molecule whose IR counterpart may remain overlapped in the broad band centered at around 948 cm -1. The other vibrational mode that has salient contribution from the stretching vibration of the thiol form of the molecule is observed at 1365 cm -1 in the NRS and in the FTIR spectra. Considerable attention can be drawn regarding the appearance of weak band in the FTIR spectra at ~ 2641 cm -1, assigned to ν(s 6 H 9 ) stretching vibration of the thiol of the molecule. The S 6 H 9 stretching vibration can be considered as the marker band, representing the propinquity of the thiol form of the molecule. Alternatively, the band at ~ 2641 cm -1 in the FTIR spectra may also be assigned as a component of the structured H- bonded N H stretching mode of the molecule [16]. Apart from the vibrational signatures contributing from the thione and the thiol forms of the molecule as discussed above, the vibrational signatures contributing from both the thione and the thiol forms of the molecule, are observed at 409, , 1045, 1082, 1156 and 1554 cm -1 in the NRS and in the FTIR spectra. Thus, from the above analysis of the Raman and the FTIR spectra, it is quite plausible to think both the thione (4-MTTN) and the thiol form (4-MTTL) of the molecule co- exist in the solid state.

157 ph dependent NRS spectra of the molecule in aqueous solution 3.6 ph Normalized intensity ph Normalized intensity ph Normalized intensity ph Normalized intensity Wavenumber / cm -1 Figure 7.3: Normalized NRS spectra of the molecule in aqueous solution (0.1 M) at varied ph. (λ exc = nm).

158 148 The ph dependent NRS spectra of the molecule at 0.1 M in aqueous solution are shown in Figure 7.3. All the spectra have been normalized with respect to 697cm -1, which appear as well resolved Raman band in the entire ph dependent NRS spectral profile. The Raman bands recorded in aqueous solution at varied ph are broadened and some of them have small blue shifts in comparison with its NRS counterpart recorded in solid state. Compared to the normalized NRS spectra of the aqueous solution of the molecule at neutral and at alkaline ph (ca. ~ ph 7 and ~ ph 9), the normalized spectra at acidic ph (ca. ph~ 2 and ph~ 4) exhibit large number of Raman bands. Weak but prominent band is observed at 525 cm -1 along with a shoulder at around 540 cm -1 in the NRS spectra of the molecule at ph~ 2 and ph~ 4. At neutral and at alkaline ph, 525 cm -1 band disappears and remain overlapped under the broad band profile of 540 cm -1 vibrational mode. The bands at 525 cm -1 and 540 cm -1 have prevailing contributions from the out-of -plane γ (S 6 C 3 N 2 H 12 ) bending and r(c 3 S 6 ) stretching vibrations respectively of the thione form of the molecule. However, the presence of both the bands in the ph dependent NRS spectra marks the existence of the thione form of the molecule at acidic, neutral and at alkaline ph. Raman band at ~ 836 cm -1, which is absent in the solid state NRS spectra but appears in the FTIR as well resolved band at around 852 cm -1, is moderately intense in the NRS of aqueous solution of the molecule at ~ph 2. The band weakens at ~ph 4 and disappears at higher ph. This band has significant contribution from the out-of-plane γ (N 2 N 1 C 5 H 8 ) bend emanating from the thione form of the molecule. The appearance of this band may signify the existence of the thione form of the molecule at acidic ph. However, it is to be mentioned that in general the out-of-plane modes show strongly in the infrared and weakly in the Raman [17-18]. Therefore, the moderately strong intensity of the band at ~ph 2, its weakening at ~ph 4 and subsequent disappearance at neutral and at alkaline ph may also be due to the protonation of the N 1 atom of the thione form of the molecule. Similar behaviors are also observed for the bands centered at around 1261 and 1609 cm -1, ascribed to in-plane α (N 1 C 5 H 8 ); α (N 2 N 1 C 5 ) bending and r (C 5 N 1 ) stretching vibrations respectively, originating from the thione form of the molecule. Both these modes are moderately intense at ph~2 and disappear or appear very weak at alkaline and at neutral ph. These vibrations not only signify the existence of the thione form of the molecule at lower ph, but also may indicate the possible protonation of the N 1 atom of the molecule. The protonation of 1, 2, 4- triazole moiety of the thione form of the molecule may result in the perturbation of vibrations involving the protonated Nitrogen (N 1 ) atom resulting in the variations of Raman band intensities of 836, 1261 and 1609 cm -1 bands.

159 149 The protonation effect may also be responsible in the variations of the Raman band intensities for 1294, 1320 and 1508 cm -1 mode. Among them, the 1294 cm -1 band is intense and sharp in the NRS at ph ~ 2 and becomes weak and broadened at higher ph, while the 1320 cm -1 band is present only at acidic ph but disappears at neutral and at alkaline ph. Both these bands are not estimated theoretically from the DFT calculations. The Raman band centred at ~ 1294 cm -1 in the ph dependent NRS spectra of the molecule has no counterpart in the NRS or FTIR spectra of the solid. However, Bougeard et al [19] reported a vibrational band at around 1280 cm -1 in the IR spectra of 1, 2, 4- Triazole molecule in the gas phase. This vibrational mode can be related to the 1294 cm -1 band observed in the ph dependent NRS spectra of the molecule. The band has been ascribed to the ring stretching vibration of the 1, 2, 4-triazole moiety of either the thione or the thiol form of the molecule. The variation in intensity of this band may be due to the protonation effect at lower ph [[19]. The 1320 cm -1 band, ascribed to the ring-stretching mode of the thione form of the molecule, is recorded only at acidic ph in the NRS spectrum [20-21]. The intensity of this band is known to be sensitive with the extent of protonation [20-21]. The appearance of 1508 cm -1 band in the NRS spectra at acidic, neutral and at alkaline ph marks the existence of both the thione and the thiol form of the molecule. It can originate from the bending vibrations related to the externally attached CH 3 group of the thiol form of the molecule or from the in plane bending of α(c 3 N 2 H 12 ), α(s 6 C 3 N 2 ) and externally attached CH 3 group of the thione form of the molecule or from both. This band is moderately intense at acidic and at neutral ph and is very intense at alkaline ph (ca ph~9). However, the variation in intensity of this band with ph may indicate the protonation effect of the N 1 atom of the thione form of the molecule. Thus, the protonation of the molecule with ph allows us to refine the vibrational assignment, which was theoretically predicted to originate from both the thione and the thiol forms of the molecule. Interesting observation can be drawn regarding the appearance of a pair of band at ~ 1345 and 1365 cm -1 in the NRS spectra of the molecule at ph ~ 2 and 4. At neutral and at alkaline ph, the 1365 cm -1 band disappears with the appearance of a well-resolved Raman band at around 1345 cm and 1365 cm -1 bands are ascribed to in-plane bending and stretching vibrations of the thione and the thiol form of the molecule respectively. The above results indicate that at acidic ph (c.a. ph ~ 2 and 4) both the thione and the thiol forms of the molecule co-exist whereas at neutral and at alkaline ph (ph ~ 7 and 9), thione form of the molecule exists. This observation however contradicts our earlier conjecture. Considerable attention can be drawn regarding the bands centered at around 957 and 964 cm -1 in the NRS spectra of the molecule at ph ~2 and 9. Intensity reversal and considerable red shift of this pair of bands are also observed at the above-mentioned ph values. However, at the intermediate ph values (ca ph ~4 and ph ~7), band ~ 952 cm -1 is

160 150 only observed with the disappearance of 964 cm -1 band. This band can be related with 957 cm -1 band, observed in the NRS spectra of the molecule at ph 2. The 952 cm -1 (calculated 947 cm -1 ) band is ascribed to in-plane bending α(h 12 N 2 C 3 ); α(n 2 N 1 C 5 ) of the thione form of the molecule. The 957 cm -1 (calculated 950 cm -1 ) and 964 cm -1 (calculated 956 cm -1 ) are also ascribed to significant contribution from the out-of-plane γ(h 9 S 6 C 3 N 4 ) and in plane bending respectively of the 1, 2, 4- triazole moiety of the thiol form of the molecule. These observations, however, indicate preponderance of the thione form of the molecule at acidic ph and the thiol form at alkaline ph. A general conclusion can be drawn regarding the appearance of the bands at ~ 697 and 1437 cm -1 in the NRS spectra at acidic, neutral and alkaline ph. All these bands have significant contributions from the bending/stretching vibrations of 1, 2, 4- triazole moiety of both the thione and the thiol forms of the molecule. The appearances of these bands in the entire ph dependent NRS spectral profile of the molecule indicate the co-existence of both the forms of the molecule in acidic, neutral and alkaline media. The above analysis of the NRS spectra of the molecule thus help us to identify the marker bands representing vibrational signatures of thione, thiol or both forms of the molecules at acidic, neutral and alkaline media. The concomitance of the Raman bands representing vibrational signatures emanating from the thione and the thiol forms of the molecules may signify the presence of both the forms of the molecule in aqueous solution. However, the respective population of the thione-thiol tautomeric forms of the molecule varies with the ph of the solution. The quantitative measure of relative population of the tautomeric forms at various ph can be estimated from the ratio of the sum of the integrated intensities of the assigned experimental bands representing vibrational signatures of thiol ( I Thiol ) and thione ( I Thione ) forms of the molecule divided Thione Thiol by the theoretically predicted sums of absolute intensities A, A of the respective bands [22]. [ Thiol] [ Thione] I Thiol Thione = Thione Thiol I A A (7.1) The results indicate that 84 % of the thione species of the molecule are prevalent at ph ~ 2. At ph ~ 4, 7 and 10, the percent of thione species of the molecule varies in the range between %. Figure 6.4 shows the bar diagram indicating the relative percentage populatation of the thione and the thiol forms of the molecule at various ph. The bar diagram as shown in Figure 7.4, indicates the abundance of the thione form of the molecule in acidic, neutral and alkaline media. This result is in accordance with the literatures concerning with the spectroscopic and structural details of organic molecules which exist in thione-thiol tautomeric equilibrium in ground state. The predominant existence of the thione form of 2, 3-dimercapto- 1, 3, 4- Thiadiazole, 2-meracpto-5-

161 151 methy, 1, 3, 4-Thiadiazole, 4, 6-dimethyl-2-Mercaptopyrimidine molecules in aqueous (polar) solution are reported elsewhere [23-25]. Figure 7.4: Bar diagram indicating the relative percentage population of the thione and the thiol forms of the molecule Interesting observations can be drawn regarding the variation in intensities of 836, 1261, 1294, 1320, 1508 and 1605 cm -1 bands observed in the ph dependent NRS spectra of the molecule. The intensity variations of the bands with ph as discussed earlier are presumed to be due to the protonation effect of the thione form of the molecule. Interestingly, all these bands though show variations in the relative intensities with ph, however, do not show any appreciable shift in their band frequencies. The red shifts of the vibrational bands of the protonated species are general observation. The optimized geometries of protonated thione (4-MTTN.H + ), mono protonated thiol [4-MTTL H + (A) and 4-MTTL H + (B)] and diprotonated thiol forms (4- MTTL.H + H + ) of the molecule are shown in Figure 7.5 (a-d). The two hydrogen atoms + H 14 + H 13 and of the diprotonated thiol form of the molecule [Figure 7.5(d)] occupy symmetrical positions one above and the other below the plane of 1, 2, 4-triazole moiety of the molecule. The N1 and N 2 atoms of the thiol form and N 1 atom of the thione form of the molecule are the probable active sites of protonation. The monoprotonation of the thiol form of the molecule can occur either through N 1 atom [4-MTTL H + (A)] or through N 2 atom [4-MTTL H + (B)] of the molecule while the deprotonation of this form of the molecule can occur through both N 1 and N 2 atoms. The N 1... H + 14, N 2 H + 13 distances in the diprotonated thiol and N 1 H + 13 / N 2 H + 13 distance in mono protonated thiol forms of the molecule are estimated to be around Å, Å and Å

162 152 respectively. As the atomic distance between the protonated hydrogen atom/atoms with the respective nitrogen atom/atoms, of the thiol form of the molecule are small, so red shifts in vibrational frequencies of the mono and diprotonated thiol forms of the molecule are expected. Table: 7.3 Calculated frequencies of different protonated models of the thione and thiol forms of the molecule using B3LYP/ 6-31G (d,p) level of theory. 4-MTTN + H + 4-MTTL + H + (A) 4-MTTL + H + (B) 4-MTTL + H + H The optimized structure of the protonated thione form (4-MTTN.H + ) of the molecule [Figure 7.5(a)] shows that the H + 13 atom is laying above the plane of 1, 2, 4- triazole moiety of the molecule with N 1... H + 13 distance is about 2.90 Å. This atomic distance is too appreciable to produce any shift in the vibrational signatures of the protonated thione form of the molecule. No significant shift in the Raman band frequencies of 836, 1261, 1294, 1320 and 1508 cm -1 bands in the ph dependent NRS spectra of the molecule may signify the probable existence of the protonated thione form

163 153 of the molecule. The vibrational frequencies, as predicted by DFT calculations for the protonated models of the thione and the thiol forms (4-MTTN.H +, 4-MTTL.H + (A), 4-MTTL.H + (B) and 4-MTTL.H + H + ) of the molecule, are shown in Table 7.3. It is clearly seen from Table-7.3 that the vibrational frequencies of the protonated thione form of the molecule are in better agreement with the experimentally observed NRS spectra recorded at ph ~2 and ph ~ 4. To substantiate our earlier conjecture, the electron distribution in the frontier orbitals of the thione, thiol and their different protonated forms of the molecule have been estimated from the DFT calculations. They are shown in Figure 7.6. (a) 4-MTTN.H + (b) 4-MTTL.H + (A) (c) 4-MTTL.H + (B) (d) 4-MTTL.H + H + Figure 7.5: The optimized molecular structure of (a) protonated thione (4-MTTN..H + ) (b) and (c) mono protonated thiol [4-MTTL H + (A) and 4-MTTL H + (B) respectively] and (d) diprotonated thiol (4-MTTL H + H + ) forms of the molecule obtained from B3LYP /6-31G (d, p) level of theory. It is clearly seen from Figure 7.6, the electron distribution in the HOMO and LUMO orbitals of 4-MTTL, 4-MTTL.H + (A), 4-MTTL H + (B) and 4-MTTL.H + H +

164 154 are remarkably different while the frontier orbitals of 4-MTTN and 4-MTTN.H + are identical. HOMO of 4-MTTN HOMO of 4-MTTN H + LUMO of 4-MTTN LUMO of 4-MTTN H + Figure 7.6(a). Calculated HOMO and LUMO orbitals of thione (4-MTTN) and protonated thione (4-MTTN..H + ))(Isocontour: 0.02 a.u.) (b) HOMO of 4-MTTL LUMO of 4-MTTL Figure 7.6(b). Calculated HOMO and LUMO orbitals of thiol (4-MTTL)(Isocontour: 0.02 a.u.)

165 155 HOMO of 4-MTTL H + (A) HOMO of 4-MTTL H + (B) HOMO of 4-MTTL H + H + LUMO of 4-MTTL H + (A) LUMO of 4-MTTL H + (B) LUMO of 4-MTTL H + H + Figure 7.6. Calculated HOMO and LUMO orbitals of mono protonated [4-MTTL H + (A), and 4- MTTL H + (B)] and diprotonated thiol (4-MTTL H + H + ) (Isocontour: 0.02 a.u.) The identical HOMO and LUMO orbitals of the thione and its protonated form of the molecule may indicate that the electronic charge density remain unperturbed upon protonation. This result further substantiates that there is indeed no change in electron distribution in the frontier orbitals of the thione and its protonated form, to produce any shift in Raman band frequencies in the ph dependent NRS spectra of the molecule ph dependent SERS Spectra of the molecule The SERS spectra of the molecule at 1.0x10-6 M adsorbate concentration for various ph values of the colloidal silver surface are shown in Figure 7.7. The spectra at various ph values are characterized by the SER bands mostly concentrated between cm -1 wave number ranges. The 1167, 1558 and 1605 cm -1 bands those are weak at acidic ph (ca. ph ~ 2 and 4) gain in intensity at neutral ph. The SERS spectrum of the molecule recorded at alkaline ph has much higher signal to noise ratio than the spectra recorded at acidic and at neutral ph.

166 156 Counts/mW/scan ph ph-7 Count/mW/scan ph-4 Counts/mW/scan ph-2 5 Count/mW/scan Wavenumber / cm -1 Figure 7.7: ph dependent SERS spectra of the molecule in silver hydrosol at 1.0x 10-6 M concentration (λ exc = nm). Considerable attention can be drawn for the band centered at ~ 1488 cm -1 in SERS spectra of the molecule at acidic, neutral and alkaline ph. This band has prevailing contribution from the in-plane bending of α(c 3 N 2 H 12 ) of the thione form of the

167 157 molecule. It is considerably red shifted by ~ 20 cm -1 in comparison to its NRS counterpart in solution. Intensity reversal is observed for the pair of bands cantered at around 1318, 1352 and 1488, 1352 cm -1. The 1352 cm -1 band, ascribed to in-plane bending vibrations of the thione form of the molecule, gains in intensity with the increase in ph. The NRS counterpart of this band is observed at ~1345 cm -1. The appearance and enhancement of 1345 cm -1 in the entire ph dependent SERS spectral profile indicates the adsorption of the thione form of the molecule with the colloidal silver surface. The N-H stretching modes as often reported in the cm -1 which can be considered to be the marker bands of the thione form of the molecule, are however not observed in the entire ph dependent SERS spectral profile, probably due to the intense broad background of the OH stretching mode (centered around 3400 cm -1 ) of bulk water. However, it is reported that the intensities of bands observed in the SERS spectra generally fall off with increasing vibrational frequency [26]. This may be considered as another explanation concerning the absence of N H stretching modes in the SERS spectra. The vibrational signatures having prevailing contribution from the adsorption of the thiol form of the molecule are not recorded. The possible adsorptive sites of the thione form of the molecule are Nitrogen (N 1 ), Sulfur (S 6 ) and the delocalized π-electron cloud of the 1, 2, 4 triazole ring moiety. Significant down shift of the 1488 cm -1 band in the entire ph dependent SER spectral profile indicate the possible interaction of the π-electron cloud of the 1, 2, 4 triazole ring moiety of the molecule at acidic, neutral and at alkaline media [27]. The adsorption through Nitrogen (N 1 ) and Sulfur (S 6 ) atoms of the molecule can be estimated theoretically by enumerating the negative charge density on each of these probable active sites [6-7,28]. The higher the negative charges density on the atom, the higher the probability of it to act as an adsorptive site for the silver substrate. Theoretical results estimated from DFT calculations show that the partial charges on the N 1 and S 6 atoms of the thione form of the molecule determined by the natural population analysis (NPA) are and , respectively. The negative charge density is thus observed to be equally appreciable on the nitrogen (N 1 ) and on sulfur (S 6 ) atom. These results may indicate active participation of both the nitrogen (N 1 ) and sulfur (S 6 ) atoms in the adsorption process. The appearance of intense but broad shoulder at ~ cm -1 in the entire ph dependent SERS spectral profile, ascribed to Ag-N and Ag-S stretching vibrations [29-30], indicates that the 1, 2, 4 triazole moiety of the thione form of the molecule is indeed adsorbed through the nitrogen (N 1 ) and sulfur (S 6 ) atoms via σ-bond formation. The adsorption through both the nitrogen (N 1 ) and sulfur (S 6 ) atoms of the thione form of the molecule may result in the enhancement of the SER bands at ~ 1168, 1352, 1558 and 1605 cm -1. The generalized two-dimensional (2D) correlation spectroscopy has been employed for further interpretation of the ph dependent SERS spectra of the molecule.

168 158 2D-correlation spectroscopy simplifies the investigation of complex spectra, enhancing spectral resolution by spreading peaks along the second dimension. This technique thus enables one to extract information that cannot be obtained from the conventional 1D spectra [31]. The synchronous and asynchronous correlation spectra are generated from the 2D analysis. The synchronous spectrum in the region from 1400 to 1300 cm -1 is shown in Figure 7.8(a). (a) Synchronous spectrum (b) Asynchronous spectrum Figure 7.8: (a) Synchronous and (b) Asynchronous 2D correlation spectrum of the molecule in the range cm -1, constructed from the ph dependent SERS spectra. A synchronous spectrum is generally symmetric with respect to the diagonal line corresponding to co-ordinates ν1=ν2. Any region of the spectrum which changes intensity to a great extent under a given perturbation will show strong auto peaks in the synchronous spectrum, while those remaining near constant develop little or no auto peaks. The presence of only prominent auto peak at 1352 cm -1 in Figure 7.8 (a) indicates that intensity of this SER band only undergoes changes to a great extend under ph perturbation. Absence of any prominent auto peaks in the synchronous spectra may indicate that apart from 1352 cm -1 band, the band intensities of other bands are not sensitive with ph variation. This intensity variation of 1352 cm -1 band results in the intensity reversal of the pair of bands at 1318, 1352 and 1488, 1352 cm -1 as we discussed earlier. Alternatively, the relative variation in intensities of these pair of bands in the ph dependent SERS spectra may also be due the protonation effect of the molecule. However, this possibility is ruled out considering the fact that the protonation site of the 4-MTTN molecule is lost due to competitive adsorption of the molecule with Ag + ions of the colloidal silver surface. The asynchronous spectrum in the region from 1400 to 1300 cm -1 is shown in Figure 7.8 (b). An asynchronous cross peak develops only if the intensities of two spectral

169 159 features change out of phase with each other. This feature is especially useful in differentiating overlapped bands arising from spectral signals of different origin. The asynchronous cross peaks appear at 1363, 1352; 1375, 1352; and 1320, 1352 cm -1. The cross peaks located at 1363, and 1352, 1320 cm -1 are ascribed to stretching and in-plane bending and stretching vibrations of the thiol and the thione form of the molecule respectively. Interestingly the 1363 cm -1 band is absent in the 1D ph dependent SERS spectra. However, distinct bands appear at ~ 1352 and 1320 cm -1 in 1D SERS spectra of the molecule (Figure 7.7) whose relative intensities also change with ph. These results may indicate that bands representing vibrational signatures of thione form of the molecule are significantly enhanced compared to the vibrations representing the thiol form of the molecule. This may connote our earlier conjecture of the adsorption of the thione form of the molecule on the colloidal silver surface at acidic, neutral and alkaline ph Orientation of the molecule on the silver surface: To have a precise idea regarding the orientation of thione form of the molecule at various ph values of the solution, we estimate the apparent enhancement factors (AEF) of some selected Raman bands using the relation we reported elsewhere [6-7,28] Accordingly, AEF = σ SERS [C NRS ] / σ NRS [C SERS ] (7.2) Where C and σ represent the concentration and the peak area of the Raman bands measured from baseline. They are shown in table 7.4. The orientation of the molecule has been estimated following the surface selection rule, as predicted by Moskovits [32] and Creighton [33]. According to this rule, the normal modes of vibrations with the polarizability derivative components perpendicular to the surface will be enhanced more. If the 4-MTTN molecule is considered to be lying in the xy plane and z is perpendicular to the molecular plane, then for the edge-on adsorption, the vibrations of the in-plane A / species spanning as xx or yy (depending upon the stance of the molecule on the colloidal silver surface) are expected to undergo significant enhancement. The least intense band should belong to the out-of-plane A // species spanning as yz and xz. It is clearly seen from table-7.4 that we obtain a moderate 4-5 orders of magnitude enhancement of all the bands principally representing the in-plane vibrations of the A / species of the molecule at various ph values of the colloidal silver surface. No significant enhancement contributing from the out-of-plane vibrations of the A // species of the molecule are recorded, though considerable red shift is observed for the 1488 cm -1 band in the SERS spectra at acidic, neutral and acidic ph.

170 160 Table 7.4: Observed SERS bands of the molecule at different ph along with selected Apparent Enhancement Factors and Probable Tensor Element SERS (NRS) ph-2 ph-4 ph-7 ph-9 AEF SERS (NRS) AEF SERS (NRS) AEF SERS (NRS) AEF P.T.E. (symm. species) (368) (496) 512(512) (664) 669(667) 667(665) (697) 2.0x 700(697) 3.56x 699 (697) 4.0x α yy (A / ) 59r 4, Tentative assignment. 33α 12,2, (1007) (1165) 1167 (1168) 1207 (1204) 1268(1273) 1272 (1266) 1315(1320) 1318 (1317) 6α 8,1,5 1168(1164) 1166( ) α yy (A / ) 42α 10,11, (1262) (.) (1345) 8.30x 1352(1345) 2.2x 1350(1342) 2.0x 1351(1349) 4.2x (1450) α 10,7,4 α yy (A / ) 47α 10,11,7 24α 12,2,3 1488(1508) 1.50x 1487(1507) 2.0x 1485(1507) 2.1x 1489( ) α yy (A / ) 74α 10,11, α 12,2,3 1558(1542) 1.80x 1560(1542) 2.2x 1559( ) 2.36x α yy (A / ) 85α 10,11, (1609) 1607(1609) 1602(1604) α yy (A / ) 81r 1,5 1623(1638) 24α 12,2,3

171 161 These results together with the appearance of broad and overlapped Ag-N, Ag-S stretching vibrations and the enhancement of SER bands involving nitrogen (N 1 ) and sulfur (S 6 ) atoms of the thione form of the molecule suggest that the molecules are adsorbed onto the nano colloidal silver surface through the N 1 and S 6 atoms with the molecular plane tilted with respect to the silver surface at acidic, neutral and alkaline ph. This type of adsorption geometry of the molecule may result in the enhancement of the normal modes of vibrations having α yy, polarizability tensor component. The relative small variation in intensities and enhancement factors of the SER bands at acidic, neutral and alkaline ph values may indicate fluxional motion of tilted 4-MTTN molecule on colloidal silver surface yielding a distribution of tilted orientations. 7.3 Conclusion The IR and Raman spectra of the 4-MTTL molecule in solid state and in aqueous solution have been recorded. The Raman spectra of 4-MTTL molecule and its thione form are also estimated theoretically using DFT. The observed vibrational bands have been assigned from the potential energy distributions (PED). The ph dependent NRS spectra of the molecule in aqueous solution reveal the protonation effect and preferential existance of the tautomeric form/ forms of the molecule in acid, neutral and alkaline media. The concomitance of the Raman bands representing vibrational signatures emanating from the thione and the thiol forms of the molecules signify the presence of both the forms of the molecule in aqueous solution. However, the respective population of the thione-thiol tautomeric forms of the molecule varies with the ph of the solution. The orientations of the adsorbed species on colloidal silver surface have been estimated using the surface selection rule from the ph dependent SERS spectra. The appearance of overlapped Ag-N, Ag-S stretching vibrations, considerable red shift of 1488 cm -1 band and enhancement of all the bands principally representing the in-plane vibrations of the A / species of the thione form of the molecule in the SERS spectra. These suggest that the molecules are adsorbed onto the nano colloidal silver surface through the lone pair electrons of N 1 and S 6 atoms with the molecular plane tilted with respect to the silver surface at acidic, neutral and alkaline ph.

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174 164

175 164 CHAPTER 8 Concentration-Dependent Orientational Changes of 2- Amino-2-thiazoline Molecule Adsorbed on Silver Nanocolloidal Surface Investigated by SERS and DFT 8.1 Introductory remarks 2-Amino-2-Thiazoline (2ATH) molecules, containing the amidine group, are not only important heterocyclic compounds in synthetic organic chemistry, but also are widely involved in enzyme-catalyzed biotransformation processes in biochemistry. Interestingly, this molecule may exist in amino and imino tautomeric forms in nature. In this chapter, the results of the investigation on the concentration-dependent SERS study of the biologically important, 2ATH molecule, adsorbed on silver nanocolloids is described and compared with its FTIR and NRS spectra in varied environments. The optimized structural parameters, preferential existence and computed vibrational frequencies of the tautomeric amino and the isomeric imino forms of the molecule in the gas phase and in methanol solvent have been estimated from the DFT calculations. The observed Raman signals along with the corresponding FTIR bands have been assigned and presented for the first time from the potential energy distributions (PED) in terms of internal coordinates of the molecule estimated from the results of DFT calculations. The adsorptive behavior and adsorption geometry of the preferred tautomeric form/forms of the molecule on nanocolloidal silver surface at different adsorbate concentrations, close to that encountered under physiological conditions in living systems have been elucidated from the SERS spectra. 8.2 Results & Discussion: Molecular structure The optimized molecular structures of the amino and imino tautomeric forms of the molecule are shown in Figure 8.1. The imino tautomeric form however can

176 165 exits in two isomeric forms, E-isomer and Z-isomer depending upon the orientation of hydrogen (H 11 ) atom in space linked to form the imine group (N 6 H 11 ) of the molecule. In order to retrieve some ideas concerning about the relative stability of different forms of the molecule in gas phase and in methanol solution, minimum energies of the molecule at their respective optimized geometries have been computed using the DFT method. (a) Energy = ( ) ev (b) Energy = ( ) ev (c) Energy = ( ) ev Figure 8.1:The optimized molecular structure and energy of the (a) amino (b) and (c) E- and Z-isomer of imino forms of the molecule in methanol solution obtained from B3LYP /6-31G (d, p) level of theory. The numbers in the parenthesis refer to the gas phase energy values of respective tautomeric forms of the molecule. The theoretical results indicate that existence of both the tautomeric forms of the molecule are thermodynamically favorable in gaseous state and in methanol solution. The corresponding energies at global minima of the potential energy surfaces of different forms of the molecule are also shown in Figure 8.1. Table 8.1 shows the selected optimized structural parameters, and rotational constants of different molecular forms.

177 166 Table 8.1: Relevant Structural Parameters and rotational constants of Amino, E-and Z- isomeric Imino forms of the molecule calculated from B 3LYP /6-31G (d, p) level of theory. Bond length (Ǻ) Amino form Imino form E-isomer Z-isomer In gas In In gas In In gas In C 1 N C 1 N C 1 S H 11 N C 4 C N 3 C Bond angle (degree) S 2 C 1 N S 2 C 1 N N 3 C 1 N C 1 S 2 C C 1 N 3 C H 7 C 4 H Dihedral angle (degree) S 2 C 5 C 4 N S 2 C 1 N 3 C C 1 S 2 C 5 C C 1 N 3 C 4 C S 2 C 1 N 6 H Rotational constant (GHz) A B C The DFT results indicate that thiazoline moiety of both the amino and the Z- isomeric (E-isomeric) imino tautomeric forms of the molecule show non-planar equilibrium configuration and adopt a half chair conformation with dihedral angle S 2 C 5 C 4 N 3 ~30.61º/30.76º and º(-38.18º)/-37.32º(-36.87º) respectively in gaseous phase/ methanol solvent. The C 1 N 3 bond length of the Z-isomeric (Eisomeric) imino form of the molecule in gaseous/ methanol solvent is considerably increased from 1.275/1.280Å to (1.394)/1.376 (1.378) Å in comparison with the C 1 N 3 bond length of amino form of the molecule in gas phase/methanol solvent. Moreover the C 1 N 6 bond length of the Z-isomeric (E-isomeric) imino form of the molecule is shortened by (0.101)/0.087 (0.086) Å compared to the amino form of the molecule in gas phase/methanol solvent. These results indicate the breaking of

178 167 one of the N-H bond of the exocyclic -NH 2 group of the amino form of the molecule and subsequent formation of single C 1 N 3 and double C 1 =N 6 bonds in the isomeric imino form of the molecule. Considerable increase in S 2 C 1 N 6, N 3 C 1 N 6, C 1 S 2 C 5 and C 1 N 3 C 4 bond angles are estimated for the isomeric imino form of the molecule compared to the amino form. Appreciable decrease in S 2 C 1 N 3 bond angle by ~ 9º of the isomeric imino form of the molecule in comparison with its tautomeric amino form is estimated. 20 (b) 678 Counts/mWatt/Scans Wavenumber/cm (a) Absorbance Wavenumber /cm -1 Figure 8.2: a) FTIR spectrum of the molecule of neat powder in KBr pellet. b) Normal Raman spectra of the molecule in solid state (λ exc = nm). Considerable variations in the dihedral angles mostly representing the planes of the thiazoline moiety of both the forms of the molecule are also observed for the gaseous phase and in methanol solution. These are also tabulated in Table 8.1. Structural differences are also estimated for the isomeric pair of the imino form of the

179 168 molecule. S 2 C 1 N 6 and N 3 C 1 N 6 bond angles of Z-isomeric form is considerably increased and decreased by ~8.03/7.02 and ~7.29/7.95 respectively compared to E- isomeric imino form of the molecule in gas phase/ methanol solvent. The nitrogen (N 6 ) atom of the exocyclic NH 2 group of the amino form of the molecule is sp 2 hybridized with the relevant bond angles ~ º/114.41º in gas phase/methanol solvent. The two pairs of hydrogen atoms H 7, H 8 and H 10, H 9 of the amino and imino forms of the molecule occupy symmetrical positions one above and the other below the plane of thiazoline moiety of both the tautomeric forms of the molecules. Recent crystallographic investigations of 2-Amino-2-Thiazoline molecules however revealed hydrogen bonded ribbon involving graph set dimer association via N-H N and N- H S interactions [1]. However, the theoretical results obtained by the DFT calculations are almost comparable with the reported structural parameters of 2- Amino-2-Thiazoline and related molecules [1,2] Normal Raman and FTIR Spectra of the molecule and their Vibrational Assignment: 2-Amino-2-Thiazoline (2ATH) molecule and its isomeric imino (2ITH) forms have 12 atoms; hence, each of them has 30 fundamental vibrations. Both the tautomeric forms of the molecule belong to the C S point group, and under this symmetry the 30 fundamental vibrations of the molecule are classified as Γ vib = 21 A / + 9 A // (8.1) Simple group theory predicts that 21 planar (A / ) and 9 nonplanar (A // ) species are expected to appear both in the Raman and in the IR spectra. However, among these vibrations originating from the tautomeric forms of the molecule, some vibrations are degenerate. The FTIR spectra of powdered molecule in KBr pellet and the NRS of the molecule in neat solid are shown in Figure 8.2a and 8.2b respectively. The NRS spectrum of the molecule at 0.1 M in methanol solution is depicted in Figure 8.3a. The simulated Raman spectra of the E-, Z-isomeric imino forms and the amino form of the molecule in methanol solvent are shown in panels a, b and c of Figure 8.3 respectively.

180 169 Raman activity (A 04 /a.m.u.) (d) 1707 Raman activity (A 04 /a.m.u.) (c) (b) Raman activity (A 04 /a.m.u.) Normalized intensity * * (a) Wavenumber /cm -1 Figure 8.3 (a) Normalized NRS spectra of the molecule in methanol solution at 0.1 M (* denote the solvent bands;λ exc = nm; normalization has been done with respect to 1032 cm -1 band of methanol solvent.). The theoretical gas-phase Raman spectrum of the (b) and (c) E-and Z-isomer of imino and (d) amino forms of the molecule calculated using B3LYP /6-31G (d, p) level of theory.

181 170 Table 8.2: Definition of internal and local symmetry coordinates of amino form of the molecule Definition of internal coordinates Definition of local symmetry coordinates R1 r (C 1 S 2 ) S1 2R13 R14 R15 N 3 -C 1 -S 2 bend R2 r (C 1 =N 3 ) S2 R14 R15 C 1 N 6 IP bend R3 r (C 1 -N 6 ) S3 R33 C 1 =N 3 OP bend R4 r (N 3 C 4 ) S4 R1 S 2 C 1 str. R5 r (C 4 C 5 ) S5 R35 S 2 C 1 tor. R6 r (S 2 C 5 ) S6 R2 C 1 =N 3 stretch R7 r(c 4 H 7 ) S7 R36 C 1 =N 3 tor. R8 r(c 4 H 8 ) S8 R3 C 1 N 6 str. R9 r(c 5 H 9 ) S9 R34 C 1 N 6 tor. R10 r(c 5 H 10 ) S10 R17 C 1 =N 3 C 4 bend R11 r(n 6 H 11 ) S11 R4 N 3 C 4 str. R12 r (N 6 H 12 ) S12 R37 N 3 C 4 tor. R13 θ (S 2 C 1 =N 3 ) S13 5R18 R19 R20 R21 R22 R23 N 3 C 4 C 5 bend R14 θ (S 2 C 1 N 6 ) S14 -R19 R20 R21 R22+4R23 H 7 C 4 H 8 scis. R15 θ (N 3 =C 1 N 6 ) S15 R19+R20 R21 R22 H 7 C 4 H 8 wag. R16 θ (C 1 S 2 C 5 ) S16 R19 R20 R21+R22 H 7 C 4 H 8 twist R17 θ (C 1 =N 3 C 4 ) S17 R19 R20+R21 R22 H 7 C 4 H 8 rock. R18 θ (N 3 C 4 C 5 ) S18 R7+R8 H 7 C 4 H 8 sym. Str. R19 θ (N 3 C 4 H 7 ) S19 R7 R8 H 7 C 4 H 8 asym. Str. R20 θ (N 3 C 4 H 8 ) S20 R5 C 4 C 5 str. R21 θ (C 5 C 4 H 7 ) S21 R38 C 4 C 5 tor. R22 θ(c 5 C 4 H 8 ) S22 5R24 R25 R26 R27 R28 R29 S 2 C 5 C 4 bend. R23 θ (H 7 C 4 H 8 ) S23 -R25 R26 R27 R28+4R29 H 9 C 5 H 10 scis. R24 θ (C 4 C 5 S 2 ) S24 R25+R26 R27 R28 H 9 C 5 H 10 wag. R25 θ (C 4 C 5 H 9 ) S25 R25 R26 R27+R28 H 9 C 5 H 10 twist R26 θ(c 4 C 5 H 10 ) S26 R25 R26+R27 R28 H 9 C 5 H 10 rock. R27 θ(s 2 C 5 H 9 ) S27 R9+R10 H 9 C 5 H 10 sym. Str. R28 θ(s 2 C 5 H 10 ) S28 R9 R10 H 9 C 5 H 10 asym. Str.

182 171 TABLE 8.2 (Continued) Definition of internal coordinates Definition of local symmetry coordinates R29 θ(h 9 C 5 H 10 ) S29 R16 C 1 S 2 C 5 bend. R30 θ(c 1 N 6 H 11 ) S30 R6 S 2 C 5 str. R31 θ(c 1 N 6 H 12 ) S31 R39 S 2 C 5 tor. R32 θ(h 11 N 6 H 12 ) S32 R32+R30+R31 H 11 N 6 H 12 sym. bend. R33 ω(c 1 =N 3, N 6, S 2 ) S33 2R32 R30 R31 H 11 N 6 H 12 asym. bend. R34 τ(s 2 C 1 N 6 H 11 ) S34 R30 R31 H 11 N 6 H 12 rock. R35 τ(n 3 C 1 S 2 C 5 ) S35 R11+R12 H 11 N 6 H 12 sym. str. R36 τ(s 2 C 1 =N 3 C 4 ) S36 R11 R12 H 11 N 6 H 12 asym. str. R37 τ(c 1 =N 3 C 4 C 5 ) R38 τ(n 3 C 4 C 5 S 2 ) R39 τ(c 1 S 2 C 5 C 4 ) r: bond length, θ: bond angle, ω: out-of-plane bending, τ: internal coordinate for torsion potential, Str: stretching, scis: scissoring, tor: torsion, wag: wagging, IP: in-plane, OP: out-of-plane, sym: symmetric, asym: asymmetric. Table 8.3: Definition of internal and local symmetry coordinates of imino form of the molecule. Definition of internal coordinates Definition of local symmetry coordinates R1 r(c 1 S 2 ) S1 2R13 R14 R15 N 3 C 1 S 2 bend R2 r(c 1 N 3 ) S2 R14 R15 C 1 =N 6 IP bend R3 r(c 1 =N 6 ) S3 R33 C 1 =N 6 OP bend R4 r(n 3 C 4 ) S4 R1 S 2 C 1 str. R5 r(c 4 C 5 ) S5 R35 S 2 C 1 tor. R6 r(s 2 C 5 ) S6 R2 N 3 C 1 stretch R7 r(c 4 H 7 ) S7 R36 N 3 C 1 tor. R8 r(c 4 H 8 ) S8 R3 C 1 =N 6 str. R9 r(c 5 H 9 ) S9 R34 N 6 H 11 OP bend. R10 r(c 5 H 10 ) S10 R17 C 1 N 3 C 4 bend R11 r(n 6 H 11 ) S11 R4 N 3 C 4 str. R12 r(n 3 H 12 ) S12 R37 N 3 C 4 tor. R13 θ(s 2 C 1 N 3 ) S13 5R18 R19 R20 R21 R22 R23 N 3 C 4 C 5 bend

183 172 Table 8.3 (Continued) Definition of internal coordinates Definition of local symmetry coordinates R14 θ(s 2 C 1 =N 6 ) S14 -R19 R20 R21 R22+4R23 H 7 C 4 H 8 scis. R15 θ(n 3 C 1 =N 6 ) S15 R19+R20 R21 R22 H 7 C 4 H 8 wag. R16 θ(c 1 S 2 C 5 ) S16 R19 R20 R21+R22 H 7 C 4 H 8 twist R17 θ(c 1 N 3 C 4 ) S17 R19 R20+R21 R22 H 7 C 4 H 8 rock. R18 θ(n 3 C 4 C 5 ) S18 R7+R8 H 7 C 4 H 8 sym. Str. R19 θ(n 3 C 4 H 7 ) S19 R7 R8 H 7 C 4 H 8 asym. Str. R20 θ(n 3 C 4 H 8 ) S20 R5 C 4 C 5 str. R21 θ(c 5 C 4 H 7 ) S21 R38 C 4 C 5 tor. R22 θ(c 5 C 4 H 8 ) S22 5R24 R25 R26 R27 R28 R29 S 2 C 5 C 4 bend. R23 θ(h 7 C 4 H 8 ) S23 -R25 R26 R27 R28+4R29 H 9 C 5 H 10 scis. R24 θ(c 4 C 5 S 2 ) S24 R25+R26 R27 R28 H 9 C 5 H 10 wag. R25 θ(c 4 C 5 H 9 ) S25 R25 R26 R27+R28 H 9 C 5 H 10 twist R26 θ(c 4 C 5 H 10 ) S26 R25 R26+R27 R28 H 9 C 5 H 10 rock. R27 θ(s 2 C 5 H 9 ) S27 R9+R10 H 9 C 5 H 10 sym. Str. R28 θ(s 2 C 5 H 10 ) S28 R9 R10 H 9 C 5 H 10 asym. Str. R29 θ(h 9 C 5 H 10 ) S29 R16 C 1 S 2 C 5 bend. R30 θ(c 1 =N 6 H 11 ) S30 R6 S 2 C 5 str. R31 θ(c 1 N 3 H 12 ) S31 R39 S 2 C 5 tor. R32 θ(c 4 N 3 H 12 ) S32 R30 N 6 H 11 IPbend. R33 ω(c 1 =N 6,N 3,S 2 ) S33 R31+R32 N 3 H 12 sym.bend. R34 ω(n 6 H 11,S 2,C 1 ) S34 R31 R32 N 3 H 12 asym.bend. R35 τ(n 3 C 1 S 2 C 5 ) S35 R11 N 6 H 11 str. R36 τ(s 2 C 1 N 3 C 4 ) S36 R12 N 3 H 12 str. R37 τ(c 1 N 3 -C 4 C 5 ) R38 τ(n 3 C 4 C 5 S 2 ) R39 τ(c 1 S 2 C 5 C 4 ) r: bond length, θ: bond angle, ω: out-of-plane bending, τ: internal coordinate for torsion potential, Str: stretching, scis: scissoring, tor: torsion, wag: wagging, IP: in-plane, OP: out-of-plane, sym: symmetric, asym: asymmetric. The primary aim of recording the NRS and FTIR spectra is to apprehend the existence of preferential tautomeric form/forms of the molecule from the assignment

184 173 of the vibrational signatures. The definition of internal and local symmetry coordinates for the amino and the imino forms of the molecule are tabulated in Table 8.2 and Table 8.3 respectively. Table 8.4 lists the experimentally observed FTIR, NRS and SERS band frequencies of the molecule. The observed vibrational signatures of the molecule are harmonized with the theoretically computed vibrational frequencies of the amino and the isomeric imino forms of the molecule in methanol solution and in gas phase. These are also shown in Table-8.4 along with their tentative assignments, as provided from the potential energy distributions (PED). The observed disagreement between the theory and the experiment could be a consequence of the anharmonicity and also may be due to the general tendency of the quantum chemical methods to overestimate the force constants at the exact equilibrium geometry [3-4]. Despite of the fact, one can see that there is a general concordance regarding the Raman band intensities as well as the position of the peaks between the experimental and calculated spectra [5-6]. The FTIR spectrum of the powdered sample and the NRS spectrum of the molecule in solid state are characterized by sharp and well-resolved vibrational bands (Figure 8.2a and 8.2b). Comparing to the NRS spectrum of the molecule recorded in solid state, the spectrum of the molecule in methanol solution, figure 8.3a exhibit large number of Raman bands. The modes arising principally from the stretching and bending vibrations of the thiazoline moiety of both the imino and the amino forms of the molecule are identified. However, it is very enigmatic to delineate the vibrational signatures emanating from the E- and Z- isomer of the imino form of the molecule. The strong and well resolved bands at around 927, 990 and 1340 cm -1 recorded in the FTIR spectra of the molecule in solid state appear weak in the NRS spectrum of the molecule in solid state and in methanol solution. The bands at ~ 927 cm -1 and at ~ 990 cm -1 have dominant contributions from the in-plane C 4 C 5 and N 3 C 4 stretching vibrations while 1340 cm -1 band has salient contribution from the H 7 C 4 H 8 wagging mode, all of them emanate from the amino form of the molecule. Interesting observations can be drawn regarding the appearance of weak but well resolved band at around 1608 cm -1 in the NRS spectra of the molecule recorded in solid state and in methanol containing eluent whose IR counterpart appears as a weak shoulder at ~ 1601 cm -1. This band has been assigned to -NH 2 asymmetric bending

185 174 vibration, which represents the characteristic frequency of the amino form of the molecule. The modes have prevailing contributions from the rocking and symmetric stretching vibrations of the exocyclic -NH 2 group of 2ATH molecule are observed as strong bands at ~ 1193 and 3436 cm -1 respectively in the NRS spectrum of the molecule in methanol solution. Among these modes, the 1193 cm -1 band is absent both in the IR and in the NRS spectrum of the molecule recorded in solid state while the 3436 cm-1 band is considerably red shifted and sharpened in the FTIR and in NRS spectra of the molecule recorded in solid state (not shown in the figure). The other vibrational signatures emanating from the amino form of the molecule are observed at ~ 355, 610 and 1232 cm -1 in the NRS spectrum of the molecule in methanol solution which have no counterpart in NRS and FTIR spectrum of the molecule recorded in solid state. The 355 and 610 cm -1 bands have significant contributions from in plane C 1 N 6 bending and S 2 C 1 stretching vibrations respectively while 1232 cm -1 band has prevailing contribution from the out-of-plane H 7 C 4 H 8 twist. These, observations may indicate the presence of the amino form of the molecule in solution and in solid state. However, the IR spectra of the molecule in the solid state and the NRS spectrum in methanol solution and in solid state are also characterized by the presence of bands, which represent vibrational signature principally originating from the isomeric imino form of the molecule. Sharp and intense bands centered at around 678 and 688 cm -1 are observed in the NRS spectrum of the molecule in solid state and in methanol solution respectively whose corresponding IR counterpart shows strong and well resolved absorption band at ~ 682 cm -1. This band has been ascribed to S 2 C 5 stretching vibrations emanating from the E- and/or Z-isomeric imino form/forms of the molecule. Moderately intense bands at around 1258 and 1436 cm -1 appear only in the FTIR spectrum, but are absent or appear as very weak signal in the NRS spectrum of the molecule recorded in solid state and in methanol solution. However, the 1436 cm -1 band may be present in the NRS spectrum of the molecule in solution which may remain obscured in the broad band envelop of the methanol solvent centered at around 1451 cm -1. The PED calculations reveal that 1258 and 1436 cm -1 bands have significant contribution from the H 9 C 5 H 10 wag; N 6 H 11 in-plane bend and H 7 C 4

186 175 H 8 wag; N 3 H 12 asymmetric bend respectively emanating from the E- and/or Z- isomeric imino form/ forms of the molecule. Medium strong shoulder is observed in the FTIR spectrum of the molecule at ~ 1212 cm -1 ascribed to out-of-plane H 7 C 4 H 8 twist vibrations contributing from the isomeric imino forms of the molecule. This band is absent in NRS spectrum of the molecule in solid state and in methanol solution. Weak but well resolved a prominent band is observed at ~ 598 cm -1 in the FTIR and NRS spectrum of the molecule in solid state whose corresponding NRS spectrum in methanol solution appears as a kink at ~ 597 cm -1. DFT results and PED calculations suggest that this band has dominant contribution either from the C 1 =N 6 out of plane bend of the E-isomer of 2ITH or from the in-plane S 2 C 1 stretching vibrations of the Z-isomer of 2ITH molecule or from both. The appearance of the vibrational signatures contributing from the stretching, wagging and bending vibrations of the isomeric imino form of the molecule in the IR and in the NRS spectrum of the molecule in solid state and in methanol solution allow us to predict the existence of 2ITH molecule in the solid state and in methanol solution. This observation however contradicts our earlier conjecture. (a) 631 (633) cm -1 of E-isomer (b) 619 (633) cm -1 of Z-isomer Figure 8.4: Cartesian displacement and calculated (B3LYP/6-31G (d, p)) vibrational modes of isomeric imino forms of the molecule. The numbers in parenthesis refer to the experimental value of the assigned band.

187 176 Table 8.4: Observed and calculated Raman, IR and SERS bands of the molecule in varied environment and their tentative assignments a FTIR NRS SERS amino imino solid soln 10-5 M 10-9 M calcd freq. PED % Calcd Freq. E-isomer (Zisomer) PED% [Z-isomer] 3545 S36 (99) 3356 (3366) S36 (99) 3424vs 3435s 3436 S35 (99) 3288 (3344) S35 (99) 3390vs 3317s 3106w 3130s 3144 S28 (-94) 3150 (3148) S28 (95) 3080 S27 (60), S19 (29), S18 (8) 3092 (3090) S19 (-62), S27 (18), S18 (18) 3052w S19 (45), S27 (-35), S18 (14) 3080 (3079) S27 (77), S19 (12), S18 (-5) 2981ms S18 (75), S19 (-23) 3007 (3000) S18 (75), S19 (24) 1703w 1707 S6 (57), S8 (-25) 1676w/sh 1685w 1681vvw 1684 (1691) S8 (-76), S6 (11) 1643vs 1658w 1647w s/sh 1608w 1604w S33 (92) 1580w vw 1504 S14 (89), S23(6) 1462w 1465ms 1475ms S23 (91), S14 (-6) 1487 (1485) S23 (94) 1436ms 1436w 1434 (1435) S15 (38), S34 (38), S8 (-8) 1390ms (1364) S34 (-30), S15 (27), S32 (-13), S11 (-7), S2 (-5) 1340vs 1340vw 1339vw 1354 S15 (74), S8 (-9) 1300s 1307vw 1302vw S8 (-21), S24 (20), S16 (19), S34 (8), S25 (-5) 1316 (1322) S24 (36), S16 (-24), S25 (8), S6 (-6), S32 (- 5) 1281vw 1279 S24 (38), S8 (17), S15 (8), S16 (6), S34 (-6)

188 177 Table 8.4 (Continued) FTIR NRS SERS amino imino solid soln 10-5 M 10-9 M calcd freq. PED % Calcd Freq. E-isomer (Zisomer) PED% [Z-isomer] 1258s 1258vw 1262vw 1264 (1266) S24 (29), S32 (25), S15 (15), S6 (10), S11 (- 5) 1232vs 1227 S16 (44), S24 (-27), S34 (-12) 1212ms 1219 (1221) S16 (55), S24 (18), S17 (5), S20 (-5) 1193ms S34 (26), S25 (22), S16 (16), S11 (-14), S8 (6) 1177vw 1173w (1176) S25 (52), S17 (11), S24 (-9), S20 (7) 1127w 1152 S25 (-36), S34 (16), S24 (-12), S17 (-9), S20 (6) 1142 (1134) S32 (33), S11 (20), S34 (-15), S6 (-14), S8 (- 5) 1031 S17 (38), S26 (25), S25 (-21), S21 (5) 1046 (1046) S17 (29), S26 (14), S25 (-11), S16 (-7), S11 (6) 1032 (1032) S32 (-14), S17 (-13), S25 (11), S11 (10), S20 (-9) 990vs 996vw 983ms 990 S11 (-33), S20 (17), S34 (-15), S6 (-7), S10 (6) 944vw 948vs 941 (940) S20 (48), S11 (18), S25 (-5), S24 (5) 927ms 922w 918w S20 (46), S13 (-10), S11 (10), S24 (5) 873 S26 (-40), S17 (13), S22 (7), S20 (-6), S11 (- 896 (895) S26 (-54), S17 (12), S22 (-6), S13 (5) 5) vw (749) S9 (83), S3 (-12) 706vw 705s 707 S30 (46), S22 (-12), S26 (-8), S17 (8), S13 (8) 704 (701) S30 (33), S22 (-11), S26 (10), S17 (-8), S13 (7) 682s 678vs 688s 665 S30 (21), S10 (14), S1 (-13), S8 (11), S2 (-9) 679 (679) S30 (33), S4 (16), S2 (-9), S6 (7), S11 (6) 638w 636w 633s S3 (62), S32 (16) 631 (619) S4 (-42), S2 (12), S33 (-11), S30 (9); [S3 (76), S9 (8)]

189 178 Table 8.4 (Continued) FTIR NRS SERS amino imino solid soln 10-5 M 10-9 M calcd freq. PED % Calcd Freq. E-isomer (Zisomer) PED% [Z-isomer] 610vs 605 S4 (-54), S2 (15), S30 (5) 598w 592vw 597ms/sh 599 (607) S3 (76), S9 (8); [ S4 (-42), S2 (12), S33 (- 11), S30 (9)] 518w/sh 509 S32 (60), S1 (-7), S3 (-5), S8 (-5) 501vw 500s 496w 493 S32 (24), S1 (14), S29 (-12), S22 (12), S8 (- 11) 494 (492) S1 (17), S22 916), S29 (-15), S13 (-11), S26 (-8) 451w (461) S33 (29), S12 (21), S7 (-19), S1 (-9), S10 (5) 435s s 390vw 400 S9 (72), S32 (-12) 391 (384) S2 (64), S4 (15) 355ms 359 S2 (-61), S4 (-8), S34 (-6), S9 (-5), S32 (5) 242vw 251 S12 (28), S7 (-28), S21 (-17) 230vw 230w 235 (239) S21 (38), S12 (-13), S31 (-11), S10 (9), S22 (7) 142vs 162w 157 S21 (28), S31 (-24), S5 (15), S12 (-8) 143 (143) S5 (35), S9 (-34), S31 (-11), S7 (-7) a vs: very strong; s: strong; ms: medium strong; w: weak; vw: very weak; vvw: very very weak; sh: shoulder; only contributions 5 are reported

190 179 Apart from the vibrational signatures contributing from the amino and the isomeric imino forms of the molecule as discussed above, the vibrational signatures contributing from both the tautomeric forms of the molecule are observed at around 500, 633 and 1465 cm -1 in the FTIR and NRS spectrum of the molecule in solid state and in methanol solution. Intense band at ~ 390 cm -1 is observed in NRS spectrum of the molecule in solid whose counterpart in solutions appears as very weak signal at ~ 392 cm -1. The vibrational signature of this band can originate either from C 1 N 6 torsion of the amino form of the molecule or from C 1 =N 6 in plane bend of the isomeric imino form of the molecule or from both. Considerable attention can be drawn regarding the assignment of the band centered at ~ 633 cm -1 in the NRS and IR spectrum of the molecule. This band has been assigned to C 1 =N 3 out of plane bend emanating from the amino form of the molecule. However, the DFT and PED calculations further suggest that this band may also represent vibrational signatures having significant contribution from S 2 C 1 stretching vibrations emanating from the E-isomer and/ or from C 1 =N 6 out of plane vibrations originating from the Z-Isomer of the imino from of the molecule. The Cartesian displacements of the above mentioned vibrations of the Z- and E- isomeric imino form of the molecule estimated from DFT calculation are shown in Figure 8.4. The band at ~ 705 cm -1 is strong in the NRS spectrum of the molecule in methanol solution but weak in solid state. This band has prevailing contribution from S 2 C 5 stretching mode emanating either from the isomeric imino or from the amino form of the molecule or from both. Thus, from the above analysis of the NRS and FTIR spectra, it is quite plausible to think both the amino (2ATH) and the imino form (2ITH) of the molecule co- exist in the solid state and in methanol solution. The vibrational analysis of the experimentally observed IR and the NRS spectra of the molecule aided by DFT calculations thus allow us to identify the marker bands representing vibrational signatures of amino, imino or both forms of the molecules in solid and in methanol solution. The concomitance of the Raman bands representing vibrational signatures emanating from the amino and the imino forms of the molecule may signify the presence of both the forms of the molecule in solid state and in methanol solution. However, the respective population of the amino-imino tautomeric forms of the molecule may vary in solid state and in methanol solution. The quantitative measure of relative population of the tautomeric forms in methanol

191 180 solution and in solid state can be estimated from the ratio of the sum of the integrated intensities of the assigned experimental bands representing vibrational signatures of amino and imino forms of the molecule divided by the theoretically predicted sums of absolute intensities of the respective bands [7]. Figure 8.5 shows the bar diagram indicating the relative percentage population of the amino and the imino forms of the molecule in solid state and in methanol solution. The results indicate that 66% of the imino species of the molecule are prevalent in solid state while 55% of the amino species of the molecule predominate in methanol solution. Figure 8.5: Bar diagram indicating the relative percentage population of the amino and the imino forms of the molecule amino imino i = amino imino Ii Aj j imino amino I j Ai j i (8.1) This result, however, is at variance with the report of Remko et al [8]. In their report, the theoretical analysis aided by Ab initio and DFT calculations reveal that the imino form of the molecule is more stabilized than amino form in polar solvent while

192 181 in gas phase the amino form of the molecule dominates. In a separate publication reported by Xue et al [9], the results of classical Monte Carlo simulation with free energy perturbation (FEP) technique of 2ATH molecule reveal similar prediction as reported by Remko et al. However, the present experimental results obtained from detailed vibrational analysis may provide serious apprehension in the theoretical estimation and understanding of the preferential tautomeric forms of the molecule particularly in polar solvent Concentration-Dependent SERS spectra of the molecule: Normalized intensity * 1167 * 1621 (c) Normalized intensity * * (f) Normalized intensity * 1168 * 1622 (b) Normalized intensity * * 1652 (e) (a) * (d) Normalized intensity * * Normalized intensity * Wavenumber / cm -1 Wavenumber/ cm -1 Figure 8.6:Normalized SERS spectra of the molecule at adsorbate concentrations (a) 1.0x10-4 M (b) 1.0x10-5 M (c) 1.0x10-6 M (d) 1.0x10-7 M (e) 1.0x10-8 M and (f) 1.0x10-9 M for λ exc = nm. (* denote the solvent bands). Considering the entire concentration dependent spectral profile [Figure 8.6 (af)], we find that the SERS spectra of the molecule adsorbed on nanocolloidal silver surface undergo remarkable variations in the overall spectral motif especially in the two ranges (ca. 1.0x x10-6 M and 1.0x x10-9 M) of adsorbate concentrations. The SERS spectra in the concentration range 1.0x x10-6 M are

193 182 characterized by broad and strongly enhanced band at ~200 cm -1 enhanced ill resolved bands in the wave number range cm -1. and weakly The enhanced and broad band at ~200 cm -1 in the SERS spectra of the molecule at the above mentioned concentrations of the adsorbate primarily suggest the involvement of sulfur (S 2 ) and/or nitrogen (N 3, N 6 ) atoms of the molecule with the nanocolloidal silver surface. The weakly enhanced bands in the higher wave number range (ca cm -1 ) represent vibrational signatures principally originating from the characteristic in-plane vibrations of the molecule. Considerable attention can be drawn for the broad and red shifted band centered at around 625 cm -1 at this range of adsorbate concentrations, whose NRS counterpart in methanol solution is observed as sharp and well resolved band at ~ 633 cm -1. The DFT calculations reveal that 633 cm -1 (625 cm -1 in SERS) band has prevailing contribution either from the C 1 =N 3 /C 1 =N 6 out-of-plane bend of the amino/z-isomeric imino tautomeric forms of the molecule or from the in-plane S 2 C 1 stretching vibration emanating from the E-isomeric imino form of the molecule or from both. The superposition and enhancement of this degenerate vibrational signature emanating either from the amino or from the isomeric imino form of the molecule or from both may result in broadening of the band in the SERS spectra [10]. Alternatively, substantial red shift and conspicuous increase in band width of ~ 625 cm -1 band in the SERS spectra may connote considerable π d type adsorbate surface interaction [11-12]. The electron donation from the π-orbital of the C 1 =N 3 /C 1 =N 6 bond/bonds of the amino and/or Z-isomeric imino form of the molecule to the d orbitals of the silver atom of the nano colloidal surface reduces the bonding electron density of the double bond and thus lowers the frequency of υ(c 1 =N 3 )/υ(c 1 =N 6 ). The electron back donation from the metal to the adsorbate π* orbital also reduces the C 1 =N 3 /C 1 =N 6 bond strength because of the increased antibonding character of π* orbital. This result may primarily signify the adsorption of the amino and/or Z- isomeric imino form of the molecule on the nanocolloidal silver surface. In order to contemplate the origin of intense 625 cm -1 band in the SERS spectra of the molecule at this range of adsorbate concentrations, the generalized twodimensional (2D) correlation spectroscopic technique has been employed. The basic concept used to build 2D correlation spectrum is the analysis of dynamic spectrum

194 183 i.e., the spectral analysis in the frequency domain of the spectral characteristics that changes in the time domain, due to external perturbations. (a) Synchronous spectrum (b) Asynchronous spectrum Figure 8.7: (a) Synchronous and (b) Asynchronous 2D correlation spectrum of the molecule in the range cm -1, obtained from the concentrations dependent SERS spectra. 2D-correlation spectroscopy simplifies the investigation of complex spectra, enhancing spectral resolution by spreading peaks along the second dimension [13]. The concept of two-dimensional correlation spectroscopy was developed by Noda and has wide popularity, particularly among vibrational spectroscopists [13, 14]. The 2D correlation spectroscopy comprises, basically, two types of correlation spectra, the synchronous [φ (ν1,ν2)] and asynchronous [ψ (ν1,ν2)] spectra. The synchronous spectra in the region 600 cm -1 to 650 cm -1 are shown in Figure 8.7a. A synchronous spectrum characterizes the similarity between the sequential variations of spectral intensities and is generally symmetric with respect to the diagonal line corresponding to co-ordinates ν1=ν2. Any region of the spectrum which changes intensity to a great extent under a given perturbation will show strong auto peaks in the synchronous spectrum, while those remaining near constant develop little or no auto peaks. The presence of only prominent auto peak at ~ 625 cm -1 in Figure 8.7a indicates that intensity of this SER band only undergoes changes to a great extent under change in adsorbate concentrations. Absence of any prominent auto peaks in the synchronous spectra may indicate that apart from 625 cm -1 band, the band intensities of other bands are not sensitive with the variation in adsorbate concentrations. The asynchronous spectrum in the region from 600 to 650 cm -1 is shown in Figure 8.7b. An