Box 8.1 Infrared Absorbance vs. Raman Scattering Spectroscopies
Infrared absorbance spectroscopy and Raman scattering spectroscopy provide largely similar
information on the vibrational modes present in the molecules studied. The wavelengths
corresponding to the relatively small energy differences involved cover principally the 2.5–15-μm
range or, expressed in wave numbers (the inverse of wavelengths), 4000–670 cm
À1
. The physical
mechanisms underlying the two types of measurements are different. In IR absorbance spectroscopy,
photons are absorbed when their energy corresponds to the difference in energy between two levels
of excitation of a given vibrational mode, for instance, the amide I band of the CONH group. Because
the exact frequencies depend on the nature of the secondary structure elements the peptide bond is part
of, the IR spectrum yields information about the organization in space of the polypeptide. In current
FTIR instruments, illumination from a broadband IR source is modulated according to wavelength
by an interferometer, from which the light is directed through the sample and into the IR detector.
A computer converts the interferogram (as a function of wavelength) to an IR spectrum (as a function
of frequency) via Fourier transformation, which considerably improves the sensitivity and signal to
noise ratio compared to the early wavelength-dispersive instruments using wavelength scanning and
diffraction gratings.
In Raman scattering spectroscopy, photons of higher energy are used, which are not
absorbed, but scattered. Most of them are scattered elastically, meaning without any loss or gain
of energy. They yield information about the size but not the internal organization of the particle.
Some photons, however, exchange with the target molecule that amount of energy that will excite
or de-excite a vibrational mode. This amount is the same as that ceded by an IR photon when
exciting that mode, but now it manifests itself as a small energy difference, and therefore wavelength difference, between incident and inelastically scattered photons. The vibrational frequencies
thus detected are identical to those observed in an IR absorbance spectrum, but, the underlying
physical mechanisms being different, the relative intensities of the bands can vary widely between
the two kinds of spectra. Indeed, IR absorbance is observed for vibrational modes which change the
dipole moment of the molecule, whereas Raman scattering is associated with modes that produce a
change in the polarizability of the molecule. Because of this, the Raman activity of a given
vibrational mode may differ markedly from its IR activity. For example, a homonuclear molecule
such as N 2 has no dipole moment either in its equilibrium position or when a stretching vibration
causes a change in the distance between the two nuclei. Thus it shows no IR absorbance. On the
other hand, the polarizability of the bond between the two atoms varies with the stretching
vibrations, and a Raman shift is therefore observed at the frequency of the vibrational mode.
Raman spectroscopy is not very sensitive, but it presents certain advantages, among which a
relative insensitivity to the presence of water, whose absorbance of IR light is one of the principal
difficulties in IR absorbance spectroscopy.
The sensitivity of Raman scattering spectroscopy is greatly increased in resonance Raman
spectroscopy (RRS), which exploits the enhancement of the intensity of Raman bands due to the
vibration of specific bonds when the incident light excites electronic transitions in their immediate
vicinity. Thus, excitation at the absorbance wavelength of a chromophore will strongly enhance the
signals due to the bonds of the chromophore and those connecting it to the protein. RRS is therefore
extremely useful to gather detailed information about the conformation of a chromophore and the
nature of the residues, atoms, and bonds involved in liganding it.
Surface-enhanced Raman spectroscopy (SERS) exploits the strong enhancement of Raman
lines that is observed when the target molecules are adsorbed onto noble metal nanoparticles,
particularly if they happen to be located in the cranks between two particles, where surface plasmon
resonance effects are strongest. When combined with the enhancement resulting from RRS, this
effect can be so strong (up to 10
14
–10
15
 as compared to conventional Raman measurements) that
the detection of single molecules becomes possible.
For more detailed information about vibrational spectroscopy, the reader is referred to
Cantor and Schimmel (1980) and Zaccai et al. (2017).
Raman spectroscopy is an alternative to IR absorbance spectroscopy. Based on a different
physical mechanism (see Box 8.1), it also provides information about vibration frequencies in the
target molecules. Raman spectroscopy by itself is a rather insensitive approach, and, as far as MP/APol
complexes are concerned, it would face the same difficulty disentangling the contributions of the
8.2 Optical Spectroscopy Studies of Amphipols and Membrane Protein/Amphipol Complexes
395
Infrared absorbance spectroscopy and Raman scattering spectroscopy provide largely similar
information on the vibrational modes present in the molecules studied. The wavelengths
corresponding to the relatively small energy differences involved cover principally the 2.5–15-μm
range or, expressed in wave numbers (the inverse of wavelengths), 4000–670 cm
À1
. The physical
mechanisms underlying the two types of measurements are different. In IR absorbance spectroscopy,
photons are absorbed when their energy corresponds to the difference in energy between two levels
of excitation of a given vibrational mode, for instance, the amide I band of the CONH group. Because
the exact frequencies depend on the nature of the secondary structure elements the peptide bond is part
of, the IR spectrum yields information about the organization in space of the polypeptide. In current
FTIR instruments, illumination from a broadband IR source is modulated according to wavelength
by an interferometer, from which the light is directed through the sample and into the IR detector.
A computer converts the interferogram (as a function of wavelength) to an IR spectrum (as a function
of frequency) via Fourier transformation, which considerably improves the sensitivity and signal to
noise ratio compared to the early wavelength-dispersive instruments using wavelength scanning and
diffraction gratings.
In Raman scattering spectroscopy, photons of higher energy are used, which are not
absorbed, but scattered. Most of them are scattered elastically, meaning without any loss or gain
of energy. They yield information about the size but not the internal organization of the particle.
Some photons, however, exchange with the target molecule that amount of energy that will excite
or de-excite a vibrational mode. This amount is the same as that ceded by an IR photon when
exciting that mode, but now it manifests itself as a small energy difference, and therefore wavelength difference, between incident and inelastically scattered photons. The vibrational frequencies
thus detected are identical to those observed in an IR absorbance spectrum, but, the underlying
physical mechanisms being different, the relative intensities of the bands can vary widely between
the two kinds of spectra. Indeed, IR absorbance is observed for vibrational modes which change the
dipole moment of the molecule, whereas Raman scattering is associated with modes that produce a
change in the polarizability of the molecule. Because of this, the Raman activity of a given
vibrational mode may differ markedly from its IR activity. For example, a homonuclear molecule
such as N 2 has no dipole moment either in its equilibrium position or when a stretching vibration
causes a change in the distance between the two nuclei. Thus it shows no IR absorbance. On the
other hand, the polarizability of the bond between the two atoms varies with the stretching
vibrations, and a Raman shift is therefore observed at the frequency of the vibrational mode.
Raman spectroscopy is not very sensitive, but it presents certain advantages, among which a
relative insensitivity to the presence of water, whose absorbance of IR light is one of the principal
difficulties in IR absorbance spectroscopy.
The sensitivity of Raman scattering spectroscopy is greatly increased in resonance Raman
spectroscopy (RRS), which exploits the enhancement of the intensity of Raman bands due to the
vibration of specific bonds when the incident light excites electronic transitions in their immediate
vicinity. Thus, excitation at the absorbance wavelength of a chromophore will strongly enhance the
signals due to the bonds of the chromophore and those connecting it to the protein. RRS is therefore
extremely useful to gather detailed information about the conformation of a chromophore and the
nature of the residues, atoms, and bonds involved in liganding it.
Surface-enhanced Raman spectroscopy (SERS) exploits the strong enhancement of Raman
lines that is observed when the target molecules are adsorbed onto noble metal nanoparticles,
particularly if they happen to be located in the cranks between two particles, where surface plasmon
resonance effects are strongest. When combined with the enhancement resulting from RRS, this
effect can be so strong (up to 10
14
–10
15
 as compared to conventional Raman measurements) that
the detection of single molecules becomes possible.
For more detailed information about vibrational spectroscopy, the reader is referred to
Cantor and Schimmel (1980) and Zaccai et al. (2017).
Raman spectroscopy is an alternative to IR absorbance spectroscopy. Based on a different
physical mechanism (see Box 8.1), it also provides information about vibration frequencies in the
target molecules. Raman spectroscopy by itself is a rather insensitive approach, and, as far as MP/APol
complexes are concerned, it would face the same difficulty disentangling the contributions of the
8.2 Optical Spectroscopy Studies of Amphipols and Membrane Protein/Amphipol Complexes
395