# Resonance Raman spectroscopy

Resonance Raman (RR) spectroscopy is a name given to Raman spectroscopy when the incident laser frequency is close in energy to an electronic transition of a compound or crystal under examination. The frequency coincidence (or resonance) can lead to greatly enhanced intensity of the Raman scattering, which facilitates the study of compounds present at low concentrations.[1]

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, then 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 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.[2]

## Overview

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. Although the technique measures the energy required to change the vibrational state of a molecule as does infrared (IR) spectroscopy, the two methods are actually complementary.

Infrared spectroscopy involves measuring the direct absorption of photons with the appropriate energy to excite molecular bond vibrations. 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.

### Applications

An advantage of Resonance Raman Spectroscopy over (normal) Raman spectroscopy is that the effect has much higher intensity. An application that illustrates this advantage is the study of the dioxygen molecule 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.[3]

### X-Ray Raman scattering

In the X-ray region, enough energy is available for making electronic transitions possible. At core level resonances, X-Ray Raman Scattering can become the dominating part of the X-ray fluorescence spectrum. This is due to the resonant behavior of the Kramers-Heisenberg formula in which the denominator is minimized for incident energies that equal a core level. This type of scattering is also known as Resonant inelastic X-ray scattering (RIXS). In the soft X-ray range, RIXS has been shown to reflect crystal field excitations, which are often hard to observe with any other technique. Application of RIXS to strongly correlated materials is of particular value for gaining knowledge about their electronic structure. For certain wide band materials such as graphite, RIXS has been shown to (nearly) conserve crystal momentum and thus has found use as a complementary bandmapping technique.

## Basic theory of Raman Scattering

Main article: 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, ${\displaystyle E}$, of a photon is

${\displaystyle E=h\nu }$

where ${\displaystyle h}$ is Planck’s constant and ${\displaystyle \nu }$ 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.

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 (${\displaystyle \Delta {\bar {\nu }}}$) between the laser light and the scattered light. A Raman spectrum is generated by plotting the intensity of the reemitted light versus ${\displaystyle \Delta {\bar {\nu }}}$. 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 ${\displaystyle \Delta {\bar {\nu }}}$. 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 (${\displaystyle \Delta {\bar {\nu }}=0}$). 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 ${\displaystyle \Delta {\bar {\nu }}}$ 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 (${\displaystyle \Delta {\bar {\nu }}}$), 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.

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.

## 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.

### Why Use Resonance Hyper Raman Over Resonance Raman Spectroscopy?

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.[4]

### Resonance Raman Spectroscopy vs. Resonance Hyper Raman

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 generally involves a more intense light beam than resonance Raman spectroscopy. However, the frequency of each photon can be dropped to achieve what would otherwise require higher frequency light. Having more photons of lower energy (longer wavelength) can reduce error. (citation needed) Additionally, resonance hyper Raman spectroscopy uses brighter lasers to compensate for using lower frequency. Using a larger number of photons carries with it certain advantages, such as the detection and analysis of more dilute concentrations of molecular components with which the laser has been tuned to interact.

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.[5]

## Other Variations of Raman spectroscopy

### Polarized Raman spectroscopy

Polarized Raman spectroscopy is a specialized type of Raman spectroscopy. While conventional Raman spectroscopy identifies chemical composition, polarized Raman spectroscopy can uncover information on the orientation and shape of the chemical bonds within the sample, as well as various bond vibration symmetries.

In polarized Raman spectroscopy the light incoming on the sample, (via the excitation laser), is polarized. The polarized light from the laser interacts with the sample and Rayleigh scattering, Stokes scattering, and anti-Stokes scattering occur before the light makes its way to the detector. On its way to the detector, the light passes through a second polarized filter. This second filter is oriented either parallel or perpendicular to the polarization of the original light beam. The first polarizer can be placed in the laser’s path between the incoming light and the sample, and the second polarizer between the light coming off of the sample and the detector. This setup allows the polarization to be adjusted by those running the experiment.

#### How it works

When the molecules in a solution interact with polarized light, the light is either transmitted unperturbed, or undergoes Rayleigh scattering, or occasionally is scattered as Raman scattering. If the vibrational mode involved in the Raman scattering process totally symmetric then the polarization of the Raman scattering will be the same as that of the incoming laser beam. In the case that the vibrational mode is not totally symmetric then the polarization of the photon will be lost (scrambled) partially or totally, which is referred to as depolarization. Hence polarized Raman spectroscopy can provide detailed information as to the symmetry labels of vibrational modes. In the solid state, polarized Raman spectroscopy can be useful in examination of single crystals as in this case Raman scattering will be dependent on the relative orientation of bonds with respect to the unit cell axes and provide information on the orientation and shape of ordered substances.

#### Characterization of bond vibration symmetry

The symmetry of a vibrational mode is calculated from the depolarization ratio; the ratio of the Raman scattering with polarization orthogonal to the incident laser and the Raman scattering with the same polarization of the incident laser:

${\displaystyle {\frac {I_{r}}{I_{u}}}}$

Where ${\displaystyle I_{r}}$ is the intensity of Raman scattering when the analyzer polarizer is rotated 90 degrees with respect to incident light's axis of polarization, and ${\displaystyle I_{u}}$ the intensity of Raman scattering when the analyzer polarizer is rotated so that is aligned with the polarization of the incident laser's polarization. When polarized light interacts with a molecule it distorts it as the molecule induces an equal and opposite effect in the plane-wave causing it to be rotated by the difference between the orientation of the molecule and the angle of polarization of the light wave. If ≥ ${\displaystyle {\frac {3}{4}}}$, then the vibrations at that frequency are “depolarized;” meaning that they do not share a common orientation.[6]

## Instrumentation

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.

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[7] 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.[8]

## References

1. ^ Drago, R.S. (1977). Physical Methods in Chemistry. Saunders. p. 152.
2. ^ Hu, Songzhou; Smith, Kevin M.; Spiro, Thomas G. (January 1996). "Assignment of Protoheme Resonance Raman Spectrum by Heme Labeling in Myoglobin". Journal of the American Chemical Society. 118 (50): 12638–46. doi:10.1021/ja962239e.
3. ^ Yoshikawa, Shinya; Shimada, Atsuhiro; Shinzawa-Itoh, Kyoko (2015). "Chapter 4, Section 3.1 Resonance Raman Analysis". In Peter M.H. Kroneck and Martha E. Sosa Torres. Sustaining Life on Planet Earth: Metalloenzymes Mastering Dioxygen and Other Chewy Gases. Metal Ions in Life Sciences. 15. Springer. pp. 89–102. doi:10.1007/978-3-319-12415-5_4.
4. ^ Hyper-Raman
5. ^ Raman: Application - Chemwiki
6. ^ What is polarised Raman spectroscopy? - HORIBA Scientific
7. ^ Smith, David C.; Spencer, Joseph H.; Sloan, Jeremy; McDonnell, Liam P.; Trewhitt, Harrison; Kashtiban, Reza J.; Faulques, Eric (2016-04-28). "Resonance Raman Spectroscopy of Extreme Nanowires and Other 1D Systems". Journal of Visualized Experiments (110). doi:10.3791/53434. ISSN 1940-087X.
8. ^ Spencer, Joseph H.; Nesbitt, John M.; Trewhitt, Harrison; Kashtiban, Reza J.; Bell, Gavin; Ivanov, Victor G.; Faulques, Eric; Sloan, Jeremy; Smith, David C. (2014-09-23). "Raman Spectroscopy of Optical Transitions and Vibrational Energies of ∼1 nm HgTe Extreme Nanowires within Single Walled Carbon Nanotubes". ACS Nano. 8 (9): 9044–9052. doi:10.1021/nn5023632. ISSN 1936-0851.