Resonance Raman Spectroscopy - Resonance Raman Spectroscopy

Resonance Raman Spectroscopy

Raman spectroscopy can be used to identify chemical compounds because the values of are indicative of different chemical species. This is because the frequencies of vibrational transitions depend on the atomic masses and the bond strengths. (Heavier atoms correspond to lower vibrational frequencies, while stronger bonds correspond to higher vibrational frequencies.) Thus, armed with a database of spectra from known compounds, one can unambiguously identify many different known chemical compounds based on a Raman spectrum. The number of vibrational modes scales with the number of atoms in a molecule, which means that the Raman spectra from large molecules will be very complicated. For example, proteins typically contain thousands of atoms and will therefore have thousands of vibrational modes. If these modes have similar energies, then the spectrum may be incredibly cluttered and complicated.

Not all vibrational transitions will be “Raman active”, i.e. some vibrational transitions will not appear in the Raman spectrum. This is because of the spectroscopic selection rules for Raman. As opposed to IR spectroscopy, where a transition can only be seen when that particular vibration causes a net change in dipole moment of the molecule, in Raman only transitions where the polarizability of the molecule changes can be observed. This is due to the fundamental difference in how IR and Raman spectroscopy access the vibrational transitions. In Raman spectroscopy, the incoming photon causes a momentary distortion of the electron distribution around a bond in a molecule, followed by reemission of the radiation as the bond returns to its normal state. This causes temporary polarization of the bond, and an induced dipole that disappears upon relaxation. In a molecule with a center of symmetry, a change in dipole is accomplished by loss of the center of symmetry, while a change in polarizability is compatible with preservation of the center of symmetry. Thus, in a centrosymmetric molecule, asymmetrical stretching and bending will be IR active and Raman inactive, while symmetrical stretching and bending will be Raman active and IR inactive. Hence, in a centrosymmetric molecule, IR and Raman spectroscopy are mutually exclusive. For molecules without a center of symmetry, each vibrational mode may be IR active, Raman active, both, or neither. Symmetrical stretches and bends, however, tend to be Raman active.

In resonance Raman spectroscopy, the energy of the incoming laser is adjusted such that it or the scattered light coincide with an electronic transition of the molecule or crystal. In most materials the incoming and outgoing electronic resonances are sufficiently broad that they can not be distinguished. So, rather than exciting the molecule to a virtual energy state, it is excited to near one of its excited electronic transitions. Since the energy of these transitions differ from one chemical species to the next, this technique did not become applicable until the advent of tunable lasers in the early 1970s. (Tunable lasers are those where the wavelength can be altered within a specific range.) When the frequency of the laser beam is tuned to be near an electronic transition (resonance), the vibrational modes associated with that particular transition exhibit a greatly increased Raman scattering intensity. This usually overwhelms Raman signals from all of the other transitions. For instance, resonance with a π-π* transition enhances stretching modes of the π-bonds involved with the transition, while the other modes remain unaffected.

This aspect of Raman spectroscopy becomes especially useful for large biomolecules with chromophores embedded in their structure. In such chromophores, the charge-transfer (CT) transitions of the metal complex generally enhance metal-ligand stretching modes, as well as some of modes associated with the ligands alone. Hence, in a biomolecule such as haemoglobin, tuning the laser to near the charge-transfer electronic transition of the iron center results in a spectrum reflecting only the stretching and bending modes associated with the tetrapyrrole-iron group. Consequently, in a molecule with thousands of vibrational modes, RR spectroscopy allows us to look at relatively few vibrational modes at a time. This reduces the complexity of the spectrum and allows for easier identification of an unknown protein. Also, if a protein has more than one chromophore, different chromophores can be studied individually if their CT bands differ in energy. In addition to identifying compounds, RR spectroscopy can also supply structural identification about chromophores in some cases.

The main advantage of RR spectroscopy over traditional Raman spectroscopy is the large increase in intensity of the peaks in question (by as much as a factor of 106). This allows RR spectra to be generated with sample concentrations as low as 10−8 M. This is in stark contrast to conventional Raman spectra, which usually requires concentrations greater than 0.01 M. Also, as previously mentioned, RR spectra usually exhibit only a few peaks, and different peaks can be selected for by targeting specific electronic transitions. The main disadvantage of RR spectroscopy is the increased risk of fluorescence and photodegradation of the sample due to the increased energy of the incoming laser light. Both of these factors can be minimized by using an infrared laser instead of visible light for non resonant Raman scattering, but not in RR where the laser must be tuned to the specific resonance, unless electronic levels of lower energy are available for the system under investigation.

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