Chapter 1. Introduction. Concepts of Raman spectroscopy

Transcription

1 Chapter 1. Introduction Concepts of Raman spectroscopy 11

2 1.1 Light scattering and the Raman effect Types of electromagnetic scattering When photons from a monochromatic light source such as a laser illuminate a material, the light can be transmitted, absorbed or scattered. In the case of scattering, the majority of this light is elastically scattered by the material with the same photon energy as the incident beam. This elastic scattering is called Rayleigh scattering for small particles and molecules, Debye scattering for particles with a size on the order of the incident wavelength, and Mie scattering in the case of even larger particles. 1 The angular distribution of the scattering by these larger particles is not uniform in all directions and generally forward scattering is favored. 1 In addition to elastic Rayleigh scattering, illuminating molecules also results in light scattering of a different color than the incident excitation beam. A small fraction of photons is scattered inelastically after a transfer of vibrational or rotational energy to the scattering molecules. Possibly the first mention of this phenomenon was in 1878, when Lommel 2 described characteristics of fluorescence which were dependent on the sample and excitation energy. The inelastic scattering was predicted as a distinct phenomenon by Smekal 3 as an analogue to the Compton effect discovered just previously in 1923, and thereafter predictions on the basis of classical electromagnetic theory were made by Kramers and Heisenberg 4, Schrödinger 5, and Dirac 6 in subsequent years. 7,8 The scattered light of a different color than the excitation beam was first observed and interpreted as an exchange of vibrational energy by C.V. Raman and K.S. Krishnan in 1928, 9,10 and was virtually simultaneously reported by Landsberg and Mandelstam. 11 The importance of the finding was recognized with the Nobel prize in Physics awarded to Raman in 1930, and henceforth the effect was named after him. 7 12

3 S 1 Rayleigh Scattering Raman Scattering Stokes Virtual state Resonance Raman Fluorescence Vibration S 1 = IR absorption S 0 Elastic Inelastic Vibration S 0 Figure 1.1. A simplified Jablonski energy level diagram depicting the different energetic processes of elastic and inelastic light scattering, infrared (IR) absorption, Resonance Raman scattering (RRS), and electronic absorption, followed by fluorescence emission. S 0 and S 1 are the electronic ground state and the first excited state, respectively. Stokes and anti-stokes Raman scattering Under ambient conditions, by far the most common mode of Raman scattering is Stokes shifted; incident photons excite a molecular vibration of the material from the ground electronic (S 0 ) and vibrational state, and photon energy is lost in the vibrational motion of this molecular bond. Hence these scattered Raman photons will have lower photon energy than the incident beam, and as a result the wavelength of the scattered light is longer. Anti-Stokes Raman scattering (not depicted in Figure 1.1), produced by inelastic scatter with a molecule that already existed in a higher vibration of the S 0 level, will result in scattered photons with a gain in energy compared to the incident beam. Anti-Stokes Raman scattering is especially rare under ambient conditions, due to the decreased population of these higher excited vibrational occupancies, as characterized by the Boltzmann distribution. 12 The difference in photon energy between the incident excitation and the Raman scattering is equivalent to the energy of a molecular vibration, as will be obvious from Figure 1.1 (wherein 13

4 the various processes relevant in vibrational spectroscopy are schematically depicted). This energy difference is distinct for each vibration in a molecule. Molecules with N atoms can in theory produce Raman scattering of 3N-6 different energies (3N-5 for linear molecules). While electromagnetic radiation is often described by wavelength (λ), this energy difference (Raman shift) in Raman spectroscopy is usually expressed in wavenumber (σ), or reciprocal wavelength (cm -1 ). The relationship between wavelength and wavenumber is σ = 1/ λ 0. 8,12 Raman shifts will always have the same value when measured on a wavenumber scale, but not on a wavelength scale. Selection rules It is important to note that not all molecular vibrations produce Raman scattering of equal intensity. In general there exists the rule of mutual exclusion for infrared (IR) and Raman spectroscopies, namely that in a molecule with a center of symmetry, a vibration that is Raman active is generally not pronounced in IR spectroscopy, and vice versa. 7 The molecular center of symmetry determines the electrical dipole moment and polarizability of the molecule; the latter is a measure of the change in electron cloud distribution of a molecule in response to an applied electric field. IR absorption by a molecule results from a direct resonance between the incident light and a vibrational mode of the molecule; the resonant interaction is created by the change in dipole moment with respect to a vibrational motion. In the case of polarizable molecules that may or may not have a permanent dipole, a temporary distortion of the electron cloud around a certain molecular bond is created by interaction with incoming electromagnetic radiation from the excitation beam, creating an induced dipole moment in the molecule, which subsequently allows for radiation emitted in the form of Rayleigh and Raman scattering. 8 Thus, in a Raman spectrum only those vibrations are observed that result in a change in polarizability. The following equation expresses the relationship of polarizability to the induced molecular electrical dipole moment, μ, where E denotes the electric field vector and α is the polarizability tensor with units of C m 2 V μ = α E Additionally the Raman scattering efficiency of a molecule can be expressed by the following relationship, where I 0 is the intensity of the incident light, ν 0 is its frequency, and Δα is the change in polarizability during the particular vibration: 14

5 Raman Intensity, I ~ I 0 ν 0 4 Δα 2 From this equation it is clear that using excitation sources of higher frequency increases the Raman intensity non-linearly, in theory by a 4 th power. In practice one must also consider the effects of this higher frequency excitation on sample photodegradation, as well as towards the creation of fluorescence interference. Thus a molecule will not only produce Raman scattering of different energies, but because of the vibrational dependence on Δα, the different energies will also have different ratios of intensity. By measuring these various energy differences a spectrum is obtained that provides molecular vibrational information unique to each type of molecule. Raman spectroscopy, the observation and measurement of Raman scattering, is a powerful analytical tool in that it provides molecular information, and in most Raman modes is non-invasive. Additionally of note is that due to their low polarizability, water molecules are not very Raman active, and do not generally interfere strongly in Raman measurements as they do in IR spectroscopy. This is particularly helpful in applications of Raman spectroscopy for the study of biomedical specimens and biological samples, which can often contain large amounts of water. 1.2 Fluorescence interference A process that cannot go unmentioned in discussions of Raman scattering is interference from fluorescence emission, also shown in Figure 1.1. When a molecule is excited by an incident beam to the S 1 state or higher a photon is absorbed. The excited molecule then rapidly loses energy until it reaches the lowest vibrational state of the S 1 level, generally with an efficiency of 100%. Fluorescence as a process occurs in the subsequent transition from the S 1 to one of the vibrational states of the electronic ground state S 0, via the emission of a photon. As a rule, this emitted photon is of a lower energy than the incident excitation photon, and its energy is otherwise also generally independent of the excitation energy. 13 While Raman scattering is considered to be an instantaneous process (occurring in the time it takes for a photon to travel one wavelength, typically a few femtoseconds), fluorescence emission rates are typically 10 8 s -1 with lifetimes generally on the order of a few nanoseconds

6 The time scale difference between these processes is an important consideration for Time- Resolved measurements. Not all molecules fluoresce, but for compounds that do show fluorescence it can be 10 6 times more probable than Raman scattering. Therefore, fluorescence emission and Raman scattering from a sample may have a great overall difference in intensity. Most samples include at least a small amount of some fluorescent species, and even minute fluorescent contaminants can greatly interfere with standard Raman spectroscopy, as fluorescence photons result in a strong background and increasing overall shot noise, masking the much weaker Raman signal. This can be overcome in part by using a deep-uv excitation wavelength, 14 since very few compounds exist that show significant fluorescence emission in that wavelength region. Furthermore, the fluorescence emission that does occur generally will be considerably Stokesshifted compared to the Raman scattering, such that it will not interfere spectrally. Another possibility is to use a NIR excitation wavelength, in which case the excitation energy is generally not sufficient to excite a molecule to the S 1 state, and thus the molecule will not emit fluorescence. However, these specific excitation wavelengths are not always suitable for measurements of every kind of sample. Alternatively one may increase the signal by using a particular excitation wavelength to resonantly excite a chromophore within a complex sample matrix (Resonance Raman Spectroscopy, RRS). 15 In this scenario, an excitation wavelength is chosen that is in the vicinity of a molecular transition, as shown in Figure 1.1, and the vibrations of the molecular bonds associated with this transition will have a selectively enhanced Raman signal. Thus, while Raman scattering usually has negligible impact on fluorescence measurements, fluorescence interference is almost always an obstacle in conventional Raman measurements, and as a consequence, many advanced Raman techniques have been developed as potential solutions to this problem. In this thesis, Time-Resolved Raman Spectroscopy (TRRS), Spatially-Offset Raman Spectroscopy (SORS), and Surface-Enhanced Raman Spectroscopy (SERS), and their combinations, are used to study systems where light scattering and fluorescence are the major obstacles to analysis. 16

7 1.3 Introduction to Time-Resolved Raman Spectroscopy (TRRS) Conventional Raman spectroscopy is an increasingly important analytical tool, providing structural information based on molecular vibrations rapidly and non-invasively. Furthermore, molecules can be studied in their natural state within a matrix or solvent. This technique is most easily applicable to transparent solids, liquids, gasses, or to bulk materials. However, in the case of studying semi-opaque materials it is not possible to focus on deeper layers, as the excitation beam is randomly scattered within the material and will produce Raman photons throughout. In applying conventional Raman detection methods to such a scenario the spectra are a mixture of Raman photons from different depths but dominated by signals from the surface, and often also by fluorescence emission, as previously mentioned. Methods for Raman spectroscopy in heterogeneous scattering systems rely on the principle that surface Raman photons and Raman photons from deeper within a sample can be discriminated on the basis of their distribution in time or space, as the latter travel through a translucent material and are diffusely scattered and therefore delayed. Time-resolved Raman spectroscopy (TRRS) techniques rely on short laser pulses and gated detection to make this discrimination. It should be noted that laser pluses should not be too short; pulses shorter than 1 ps are less monochromatic, which will result in serious loss of spectral resolution. In a backscattering collection geometry, deeper Raman photons arrive at the detector later, and one can selectively collect these photons by delaying the opening of a short detector gate. The width of the detector gate is a critical aspect in TRRS measurements as it determines the temporal and depth resolution of the measurements within a sample, as well as the efficiency of fluorescence rejection. Various methods of performing TRRS measurements have been established over the years, including a stimulated Raman scattering (SRS) amplifier, 16 a streak camera, 17 a fast-gated photo multiplier tube (PMT), 18 a Kerr gate, 19 and in the case of the TRRS measurements in this thesis work, via an intensified charge coupled device (ICCD) camera. 20 The TRRS system used in this thesis is described in further detail in Chapter References 1 Ingle, J.D. and Crouch, S.R. Spectrochemical Analysis. Prentice Hall, Inc. New Jersey, 1988, Pg Lommel, E. and Wiedemanns. Annalen der Physik und Chemie, 1878, 3:251 17

8 3 Smekal, A. Zur Quantentheorie der Dispersion. Naturwiss., 1923, 11(42): Kramers, H.A. and Heisenberg, W. Über die Streuung von Strahlung durch Atome. Z. Physik, 1925, 31(1): Schrodinger, E. Quantisierung als Eigenwertproblem (Vierte Mitteilung), Ann. Phys. 1926, 81: Dirac, P.A.M. The quantum theory of dispersion, Proc. Roy. Soc. London A, 1927, 114: Graselli, J.G. and Bulkin, J.B. Analytical Raman Spectroscopy, Volume 114, John Wiley & Sons, Inc. New York, Lewis I.R., and Edwards, H.G.M. Handbook of Raman Spectroscopy; from the research laboratory to the process line. Marcel Dekker, Inc. 2001, Pgs Raman, C.V. A change of wave-length in light scattering. Nature, 1928, 121(3051): Raman, C.V. and Krishnan, K.S. A new type of secondary radiation. Nature, 1928, 121(3048): Landsberg, G. and Mandelstam, L. Über die Lichtzerstreuung in Kristallen. Naturwiss., 1928, 16: Smith, E. and Dent, G. Modern Raman Spectroscopy- A practical approach. John Wiley & Sons, West Sussex, England, 2005, pgs Lakowicz, J.R. Principles of Fluorescence Spectroscopy, second edition. Kluwer Academic/Plenum Publishers New York, 1999, pgs Asher, S. A.UV resonance Raman spectroscopy for analytical, physical, and biophysical chemistry. Anal. Chem., 1993, 65 (2): 59A 66A and 201A 210A. 15 Efremov, E.V., Ariese, F., Gooijer, C. Achievements in resonance Raman spectroscopyreview of a technique with a distinct analytical chemistry potential. Analytica Chimica Acta, 2008, 606: Duncan, M.D., Mahon, R., Tankersley, L.L., and Reintjes, J. Time-gated imaging through scattering media using stimulated Raman amplification. Optics Letters, 1991, 16(23): Tahara, T. and Hamaguchi H.O. Picosecond Raman Spectroscopy Using a Streak Camera Appl. Spectrosc., 1993, 47(4):

9 18 Van Duyne, R.P., Jeanmaire, D.L., and Shriver, D. F. Mode-Locked Laser Raman Spectroscopy-A New Technique for the Rejection of Interfering Background Luminescence Signals. Anal. Chem., 1974, 46(2): Matousek, P., Towrie, M., Stanley, A., and Parker, A.W. Efficient rejection of fluorescence from Raman spectra using picosecond Kerr gating. Appl. Spectrosc., 1999, 53(12): Efremov, E.V., Buijs, J.B., Gooijer, C., and Ariese, F. Fluorescence rejection in resonance Raman spectroscopy using a picosecond-gated intensified charge-coupled device camera. Appl. Spectrosc., 2007, 61(6):