4.6 Raman Spectroscopy R.H. ATALLA, U.P. AGARWAL, and J.S. BOND 4.6.1 Introduction Raman scattering was first observed in 1928 and was used to investigate the vibrational states of many molecules in the 1930s. Initially, spectroscopic methods based on the phenomenon were used in research on the structure of relatively simple molecules. Over the past 20 years, however, the development of laser sources and new generations of monochromators and detectors has made possible the application of Raman spectroscopy to the solution of many problems of technological interest. In many industrial laboratories, Raman spectroscopy is routinely used, together with infrared spectroscopy, for acquisition of vibrational spectra. Raman spectrometer systems for routine analytical applications are com- mercially available. An important expansion of the potential of the technique has arisen from the use of the microprobe, which permits acquisition of spectra from domains as small as one micron. Application of Raman spectroscopy to lignin analytical chemistry is rela- tively new, and only limited information has been obtained. However, the technique offers several potential advantages and, though it is complementary to infrared spectroscopy, it gives information that is not accessible with the latter alone. 4.6.2 Principle The phenomena underlying Raman spectroscopy can be described by com- parison with infrared spectroscopy as shown schematically in Fig. 4.6.1. The primary event in infrared absorption is the transition of a molecule from a ground state (M) to a vibrationally excited state (M*) by absorption of an infrared photon with energy equal to the difference between the energies of the ground and the excited states. The reverse process, infrared emission, occurs when a molecule in the excited state (M*) emits a photon during the transition to a ground state (M). In infrared spectroscopy, one derives information by measuring the frequencies of infrared photons that a molecule absorbs and interpreting these frequencies in terms of the characteristic vibrational motions of the molecule. In complex molecules, some of the frequencies are associated with functional groups that have characteristic localized modes of vibration. Springer Series in Wood Science Methods in Lignin Chemistry (Edited by S.Y. Lin and C.W. Dence) Springer-Verlag Berlin Heidelberg 1992 Raman Spectroscopy 163 As is also shown in Fig.4.6.1, the same transitions between molecular vibrational states (M) and (M*) can result in Raman scattering. A key difference between the Raman and infrared processes is that, in the former process, the photons involved are not absorbed or emitted but rather shifted in frequency by an amount corresponding to the energy of the particular vibrational transition. In the Stokes process, which is the parallel of absorption, the scattered photons are shifted to lower frequencies as the molecules abstract energy from the exciting photons; in the anti-Stokes process,which is parallel to emission, tte scattered photons are shifted to higher frequencies as they pick up the energy released by the molecules in the course of transitions to the ground state. In addition, a substantial number of the scattered photons are not shifted in frequency. The process which gives rise to these photons is known as Rayleigh scattering. This scattering arises from density variations and optical hetero- genieties and is many orders of magnitude more intense than Raman scattering. Figure 4.6.1 depicts another major difference between Raman scattering and infrared processes. To be active in the infrared spectra, transitions must have a change in the molecular dipole associated with them. For Raman activity, in contrast, the change has to be in the polarizability of the molecule. These two molecular characteristics are qualitatively inversely related. A Raman spectrum is obtained by exposure of a sample to a monochromatic source of exciting photons and measurement of the frequencies of the scattered light. Because the intensity of the Raman scattered component is much lower than the Rayleigh scattered component, a highly selective monochromator and a very sensitive detector are required. 164 R.H. ATALLA et al. The exciting photons are typically of much higher energies than those of the fundamental vibrations of most chemical bonds or systems of bonds, usually by a factor ranging from about 6 for O—H and C—H bonds to about 200 for bonds between very heavy atoms, as for example in 12. The 514.5 and 488 nm lines from an argon ion laser are often used as exciting frequencies. 4.6.3 Method 4.6.3.1 Raman System A typical Raman system consists of the following basic components: (1) an excitation source, usually a laser; (2) optics for sample illumination; (3) a double or triple monochromator; and (4) a signal processing system consisting of a detector, an amplifier, and an output device. A diagram showing various com- ponents of the Raman spectrometer is shown in Fig. 4.6.2. A number of stages are involved in the acquisition of Raman spectrum. A sample is mounted in the sample chamber and laser light is focused on it with the help of a lens. Generally, liquids and solids are sampled in a Pyrex capillary tube. The scattered light is collected using another lens and is focused at the entrance slit of the monochromator. Monochromator slit widths are set for desired spectral resolution.The monochromator effectively rejects stray light and serves as a dispersing element for incoming radiation. The light leaving the exit slit of the monochromator is collected and focused on the surface of a detector. This optical signal is converted to an electrical signal within the detector and further manipulated using detector electronics. Such a signal is stored in computer memory for each predetermined frequency interval. in a Raman Spectroscopy 165 conventional Raman system using a photomultiplier tube (PMT) detector, light intensity at various frequencies is meusured by scanning the monochromator. A plot of signal intensity against wavenumber constitutes it Raman spectrum. There are various ways in which Raman systems can be designed depending upon the nature of the information desired. They can be classified as macro- and micro-systems according to whether the information is to represent the sample at the macro level or at a particular micro-domain of the sample. For homogeneous samples, information from the two systems is identical. In con- trast, this information is different for heterogeneous samples. For exumple, a spectrum acquired over a large number of woody tritcheids indicates an aver- age over-all morphological feature and is typical of spectra acquired in the macromode. On the other hand, spectra characteristic of the middle lamella, the secondary wall, or other morphologically distinct features of woody tissue require the microsampling mode. Raman spectra obtained by using both types of systems will be considered. It is also possible to carry out time-resolved measurements of Raman spectra. This has been greatly facilitated by the avail- ability of pulsed lasers and gatable multichannel detectors. The macrumode spectra described here are acquired with an Instruments SA Jobin Yvon Ramanor HG.2S system. Sample excitation is done with either argon or krypton ion lasers. This scanning spectrometer has a thermoelectrically cooled PMT detector and is fitted with a modified Nachet 400 microscope accessory for Raman microprobe work. The microprobe is capable of providing information from domains as small as 1 µ in diameter. Recent advances in solid state detectors have led to more efficient laser Raman spectrometers. These spectrometers are based on multichannel detectors (MCD) and are at least an order of magnitude faster than the systems based on PMT detector. However, at present these systems do not match the resolution capabilities of the scanning systems. The Spex Triplemate 1877 A is a new generation instrument which can be fitted with an intensified diode array consist- ing of 1024 elements. Figure 4.6.3 shows spectra obtained with these two types of detectors. 4.6.3.2 Scattering Geometries In a Raman experiment. light scattered from a laser excited sample is collected and analyzed. Focusing lenses are used both for sample excitation and light collection purposes. Generally, there are two geometries in which a sample can be studied (Fig. 4.6.4). In the 90° geometry. the laser beam direction and the axis of the collection lens are at 90° to each other, but in the backscattering or the 180° scattering geometry they are coincident. The 90-degree scattering geometry is frequently used in the macromode work, whereas back scattering geometry is usually the choice for microprobe experiments. Specimen sampling needs are somewhat different for these two types of geometries. 166 R.H. ATALLA et al. 4.6.3.3 Specimens and Sampling Laser-induced fluorescence from lignin-containing samples is a major hindrance to obtaining reasonably good spectra.Certain lignin structural units and/or lignin radicals are evidently the major contributors to the problem. Two sampling procedures are effective in significantly reducing the fluorescence of lignocellulosics. Water lmmersion Technique The technique of water immersion involves sampling lignocellulosics in an aqueous environment. Although originally developed for Raman microprobe investigations of woody tissue,this technique has given much better quality spectra in the conventional macromode studies of lignin-containing samples. The sampling procedure for microprobe studies of woody tissue is briefly described below. Raman Spectroscopy 167 Sections of woody tissue, 30 µ in thickness, are extracted with a toluene/ ethanol mixture (2:1, v/v) and repeatedly washed with distilled water. A flat- base Pyrex glass beaker is attached to a slide in a manner that allows transmitted light to pass through. On the inner side of the bottom, another portion of a glass slide is attached. A wet cell wall section is sandwiched between the slide portion and a cover glass through the center of which a small hole has been drilled; the cover glass holds the section immobile while D2O is slowly added to the beaker until the sample is completely immersed.