Resonance Raman spectroscopy

Resonance Raman spectroscopy (RR spectroscopy) is a Raman spectroscopy technique in which the incident photon energy is close in energy to an electronic transition of a compound or material under examination. The frequency coincidence (or resonance) can lead to greatly enhanced intensity of the Raman scattering, which facilitates the study of chemical compounds present at low concentrations. Resonance Raman spectroscopy (RR spectroscopy) is a Raman spectroscopy technique in which the incident photon energy is close in energy to an electronic transition of a compound or material under examination. The frequency coincidence (or resonance) can lead to greatly enhanced intensity of the Raman scattering, which facilitates the study of chemical compounds present at low concentrations. Raman scattering is usually extremely weak, of the order of 1 in 10 million photons that hit a sample are scattered with the loss (Stokes) or gain (anti-Stokes) of energy because of changes in vibrational energy of the molecules in the sample. Resonance enhancement of Raman scattering requires that the wavelength of the laser used is close to that of an electronic transition. In larger molecules the change in electron density can be largely confined to one part of the molecule, a chromophore, and in these cases the Raman bands that are enhanced are primarily from those parts of the molecule in which the electronic transition leads to a change in bond length or force constant in the excited state of the chromophore. For large molecules such as proteins, this selectivity helps to identify the observed bands as originating from vibrational modes of specific parts of the molecule or protein, such as the heme unit within myoglobin. Raman spectroscopy and RR spectroscopy provide information about the vibrations of molecules, and can also be used for identifying unknown substances. RR spectroscopy has found wide application to the analysis of bioinorganic molecules. The technique measures the energy required to change the vibrational state of a molecule as does infrared (IR) spectroscopy. The mechanism and selection rules are different in each technique, however, band positions are identical and therefore the two methods provide complementary information. Infrared spectroscopy involves measuring the direct absorption of photons with the appropriate energy to excite molecular bond vibrational modes and phonons. The wavelengths of these photons lie in the infrared region of the spectrum, hence the name of the technique. Raman spectroscopy measures the excitation of bond vibrations by an inelastic scattering process, in which the incident photons are more energetic (usually in the visible, ultraviolet or even X-ray region) and lose (or gain in the case of anti-Stokes Raman scattering) only part of their energy to the sample. The two methods are complementary because some vibrational transitions that are observed in IR spectroscopy are not observed in Raman spectroscopy, and vice versa. RR spectroscopy is an extension of conventional Raman spectroscopy that can provide increased sensitivity to specific (colored) compounds that are present at low (micro to millimolar) in an otherwise complex mixture of compounds. An advantage of resonance Raman spectroscopy over (normal) Raman spectroscopy is that the intensity of bands can be increased by several orders of magnitude. An application that illustrates this advantage is the study of the dioxygen unit in cytochrome c oxidase. Identification of the band associated with the O–O stretching vibration was confirmed by using 18O–16O and 16O–16O isotopologues. The frequencies of molecular vibrations range from less than 1012 to approximately 1014 Hz. These frequencies correspond to radiation in the infrared (IR) region of the electromagnetic spectrum. At any given instant, each molecule in a sample has a certain amount of vibrational energy. However, the amount of vibrational energy that a molecule has continually changes due to collisions and other interactions with other molecules in the sample. At room temperature, most molecules are in the lowest energy state—known as the ground state. A few molecules are in higher energy states—known as excited states. The fraction of molecules occupying a given vibrational mode at a given temperature can be calculated using the Boltzmann distribution. Performing such a calculation shows that, for relatively low temperatures (such as those used for most routine spectroscopy), most of the molecules occupy the ground vibrational state. Such a molecule can be excited to a higher vibrational mode through the direct absorption of a photon of the appropriate energy. This is the mechanism by which IR spectroscopy operates: infrared radiation is passed through the sample, and the intensity of the transmitted light is compared with that of the incident light. A reduction in intensity at a given wavelength of light indicates the absorption of energy by a vibrational transition. The energy, E {displaystyle E} , of a photon is where h {displaystyle h} is Planck's constant and ν {displaystyle u } is the frequency of the radiation. Thus, the energy required for such transition may be calculated if the frequency of the incident radiation is known. It is also possible to observe molecular vibrations by an inelastic scattering process. In inelastic scattering, an absorbed photon is reemitted with lower energy. In Raman scattering, the difference in energy between the absorbed and reemitted photons corresponds to the energy required to excite a molecule to a higher vibrational mode.