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.
Overview
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.
Basic theory
Rayleigh scattering, Stokes Raman scattering, and anti-Stokes Raman scattering
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, , of a photon is
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.
Typically, in Raman spectroscopy high intensity laser radiation with wavelengths in either the visible or near-infrared regions of the spectrum is passed through a sample. Photons from the laser beam are absorbed by the molecules, exciting them to a virtual energy state. If the molecules relax back to the vibrational state that they started in, the reemitted photon has the same energy as the original photon. This leads to scattering of the laser light, but with no change in energy between the incoming photons and the reemitted/scattered photons. This type of scattering is known as Rayleigh scattering.
However, it is possible for the molecules to relax back to a vibrational state that is higher in energy than the state they started in. In this case, the original photon and the reemitted photon differ in energy by the amount required to vibrationally excite the molecule. Generally, the difference in energy is recorded as the difference in wavenumber () between the laser light and the scattered light. A Raman spectrum is generated by plotting the intensity of the reemitted light versus . In this example the reemitted radiation is lower in energy than the incident laser light. Consequently, the change in wavenumber is positive and results in a series of peaks in the Raman spectrum known as Stokes lines.
A Raman spectrum also exhibits peaks that correspond to negative values of . These peaks are due to reemitted photons that are higher in energy than the incident photons. This occurs when molecules that initially are in an excited vibrational state absorb the laser light and relax back to the lower vibrational state when they reemit the photon. These lines in the Raman spectrum are known as anti-Stokes lines. Since the Stokes lines and anti-Stokes lines gain and lose the same amount of energy, they are symmetric with respect to the peak due to elastic (Rayleigh) scattering (). The anti-Stokes lines are appreciably less intense than the corresponding Stokes lines. This is because initially very few molecules are in excited vibrational states compared to the number in the ground state. Since anti-Stokes lines arise from the former and Stokes lines arise from the latter, the Stokes lines are much more intense. However, in molecules that exhibit fluorescence, the Stokes lines may be obscured while the anti-Stokes lines remain unaffected. In such cases, it is necessary to use the anti-Stokes lines despite their lower intensity.
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 is complicated. For example, proteins typically contain thousands of atoms and 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 are Raman active, i.e., some vibrational transitions do not appear in the Raman spectrum. This is because of the spectroscopic selection rules for Raman spectra. 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 spectroscopy 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 are IR active and Raman inactive, while symmetrical stretching and bending is 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.
Resonance Raman scattering
In resonance Raman spectroscopy, the wavelength of the incoming laser is selected to coincide with an electronic transition of the molecule or material. Since the energy of electronic transitions (i.e. the color) varies widely from compound to compound, wavelength-tunable lasers, which appeared in the early 1970s, are useful as they can be tuned to coincide with an electronic transition (resonance), however, the broadness of electronic transitions means that multi-line lasers (Argon and Krypton ion) are commonly used. The vibrational modes that undergo a change in bond length and/or force constant during the electronic excitation can show a large increase in polarisability and hence Raman intensity. The increase can be by a factor of 10 to > 100,000 and is most apparent in the case of π-π* transitions and least for metal centered (d–d) transitions.The selective enhancement of the Raman scattering from specific modes under resonance conditions means that resonance Raman spectroscopy is 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 hemoglobin, 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 non-resonant Raman spectroscopy is the large increase in intensity of the bands in question (by as much as a factor of 106). This allows RR spectra to be obtained with sample concentrations as low as 10−8 M. This is in stark contrast to non-resonant Raman spectra, which usually requires concentrations greater than 0.01 M. RR spectra usually exhibit fewer bands than the non resonant Raman spectrum of a compound, and the enhancement seen for each band can vary depending on the electronic transitions with which the laser is resonant. Since, typically RR spectroscopy are obtained with lasers at visible and near-UV wavelengths, spectra are more likely to be affected by fluorescence. Furthermore photodegradation (photobleaching) and heating of the sample can occur as the sample also absorbs the excitation light, dissipating the energy as heat.
Instrumentation
Raman microscope
The instrumentation used for resonance Raman spectroscopy is identical to that used for Raman spectroscopy; specifically, a highly monochromatic light source (a laser), with an emission wavelength in either the near-infrared, visible, or near-ultraviolet region of the spectrum. The essential point is that the wavelength of the laser emission is coincident with an electronic absorption band of the compound of interest. The spectra obtained contain non-resonant Raman scattering of the matrix (e.g., solvent) also.
Sample handling in Raman spectroscopy offers considerable advantages over FTIR spectroscopy in that glass can be used for windows, lenses, and other optical components. A further advantage is that whereas water absorbs strongly in the infrared region, which limits the pathlengths that can be used and masking large region of the spectrum, the intensity of Raman scattering from water is usually weak and direct absorption interferes only when near-infrared lasers (e.g., 1064 nm) are used. Therefore, water is an ideal solvent. However, since the laser is focused to a relatively small spot size, rapid heating of samples can occur. When resonance Raman spectra are recorded, however, sample heating and photo-bleaching can cause damage and a change to the Raman spectrum obtained. Furthermore, if the absorbance of the sample is high (> OD 2) over the wavelength range in which the Raman spectrum is recorded then inner-filter effects (reabsorption of the Raman scattering by the sample) can decrease signal intensity dramatically. Typically, the sample is placed into a tube, which can then be spun to decrease the sample’s exposure to the laser light, and reduce the effects of photodegradation. Gaseous, liquid, and solid samples can all be analyzed using RR spectroscopy.
Although scattered light leaves the sample in all directions the collection of the scattered light is achieved only over a relatively small solid angle by a lens and directed to the spectrograph and CCD detector. The laser beam can be at any angle with respect to the optical axis used to collect Raman scattering. In free space systems the laser path is typically at an angle of 180° or 135° (a so-called back scattering arrangement). The 180° arrangement is typically used in microscopes and fiber optic based Raman probes. Other arrangements involve the laser passing at 90° with respect to the optical axis. Detection angles of 90° and 0° are less frequently used.
The collected scattered radiation is focused into a spectrograph, in which the light is first collimated and then dispersed by a diffraction grating and refocused onto a CCD camera. The entire spectrum is recorded simultaneously and multiple scans can be acquired in a short period of time, which can increase the signal-to-noise ratio of the spectrum through averaging. Use of this (or equivalent) equipment and following an appropriate protocol[5] can yield better than 10% repeatability in absolute measurements for the rate of Raman scattering. This can be useful with resonance Raman for accurately determining optical transitions in structures with strong Van Hove singularities.
Resonance hyper-Raman spectroscopy
Resonance hyper-Raman spectroscopy is a variation on resonance Raman spectroscopy in which the aim is to achieve an excitation to a particular energy level in the target molecule of the sample by a phenomenon known as two-photon absorption. In two-photon absorption, two photons are simultaneously absorbed into a molecule. When that molecule relaxes from this excited state to its ground state, only one photon is emitted. This is a type of fluorescence.In resonance Raman spectroscopy, certain parts of molecules can be targeted by adjusting the wavelength of the incident laser beam to the “color” (energy between two desired electron quantum levels) of the part of the molecule that is being studied. This is known as resonance fluorescence, hence the addition of the term “resonance” to the name “Raman spectroscopy”. Some excited states can be achieved via single or double photon absorption. In these cases however, the use of double photon excitation can be used to attain more information about these excited states than would a single photon absorption. There are some limitations and consequences to both resonance Raman and resonance hyper Raman spectroscopy.
Both resonance Raman and resonance hyper Raman spectroscopy employ a tunable laser. The wavelength of a tunable laser can be adjusted by the operator to wavelengths within a particular range. This frequency range however is dependent on the laser’s design. Regular resonance Raman spectroscopy therefore is only sensitive to the electron energy transitions that match that of the laser used in the experiment. The molecular parts that can be studied by normal resonance Raman spectroscopy is therefore limited to those bonds that happen to have a “color” that fits somewhere into the spectrum of “colors” to which the laser used in that particular device can be tuned. Resonance hyper Raman spectroscopy on the other hand can excite atoms to emit light at wavelengths outside the laser’s tunable range, thus expanding the range of possible components of a molecule that can be excited and therefore studied.
Resonance hyper Raman spectroscopy is one of the types of “non-linear” Raman spectroscopy. In linear Raman spectroscopy, the amount of energy that goes into the excitation of an atom is the same amount that leaves the electron cloud of that atom when a photon is emitted and the electron cloud relaxes back down to its ground state. The term non-linear signifies reduced emission energy compared to input energy. In other words, the energy into the system no longer matches the energy out of the system. This is due to the fact that the energy input in hyper-Raman spectroscopy is much larger than that of typical Raman spectroscopy. Non-linear Raman spectroscopy tends to be more sensitive than conventional Raman spectroscopy. Additionally, it can significantly reduce, or even eliminate the effects of fluorescence.
