356
H. J. Byrne et al.
absorption bands with molecular structures [3]. Coblentz helped establish IR spectroscopy as a routine analytical tool, cataloguing the spectra of hundreds of substances, both organic and inorganic [3]. technological developments post world
war II aided considerably in establishing IR spectroscopy as a routine laboratory
characterisation technique, but none more so than the development of commercial
Fourier transform IR (FtIR) spectrometers in the 1960s and 1970s [4, 5] and FtIR
microscopes in the late 1980s [6].
IR spectroscopy is now a routine technique for materials characterisation and has
found numerous applications in forensics [7], environmental science [8] and pharmacology [9]. Raman spectroscopy is a complementary technique with origin in the
discovery of the Raman effect in 1928 [10], for which C.v. Raman was awarded the
Nobel prize in physics in 1930. In 1998 the Raman Effect was designated an ACS
National historic Chemical Landmark, in recognition of its importance in materials
and process analysis. Raman spectroscopy remained largely a curiosity until the advent of the laser in the 1960s, and the revolution in Charged Coupled detector (CCd)
arrays in the 1980s and 1990’s added to the benefits of high laser source intensities.
In addition, the development of narrow band laser line rejection filters meant that the
huge losses in signal from traditional triple monochromator systems could be overcome with the combination of a filter set and a single spectroscopic grating. Furthermore, the significant reductions in acquisition time with multichannel signal detection enabled significant improvements in signal to noise ratios [11]. the combination
of technology developments led to a new range of Raman spectroscopic microscopes
in the 1990s, establishing Raman spectroscopic microscopy as a relatively inexpensive benchtop laboratory tool to rival conventional infrared spectroscopy.
Similar to IR spectroscopy, Raman spectroscopy entails the coupling of incident
radiation with molecular vibrations and the resultant Raman spectrum is characteristic of the compound or material. however, whereas IR spectroscopy involves the
absorption of radiation, Raman spectroscopy is a scattering technique, whereby the
incident radiation couples with the vibrating polarisation of the molecule and thus
generates or annihilates a vibration. the differing underlying mechanisms gives
rise to a complementarity of the two techniques. For a vibration to be active in IR
spectroscopy, a change in dipole is required, whereas to be Raman active, a change
in polarisability is required. vibrations of asymmetric, polar bonds thus tend to be
strong in IR spectra, whereas Raman is particularly suitable as a probe of symmetric, nonpolar groups. Notably, oh vibrations of water are very strong in IR spectra,
whereas they are extremely weak in Raman spectra, rendering Raman a potentially
more suitable technique for bioapplications.
A further implication of the differing physical origins of the techniques is that
whereas IR monitors the absorption of IR radiation, Raman scattering can be employed in the uv, visible or near IR regions of the spectrum. Raman scattering thus offers intrinsically higher spatial resolution for mapping or profiling, the diffraction limit
being determined by the wavelength (< 1 µm for Raman, ~ 5–10 µm for IR). For many
applications, however, near IR is favoured as a source for Raman analysis, to minimise
interference from scattering, fluorescence, or photodegradation of the sample [12].
FtIR has become an accepted tool in biophysics for analysis of the structure
and interactions of proteins [13], lipids [14], carbohydrates [15] and nucleic acids
H. J. Byrne et al.
absorption bands with molecular structures [3]. Coblentz helped establish IR spectroscopy as a routine analytical tool, cataloguing the spectra of hundreds of substances, both organic and inorganic [3]. technological developments post world
war II aided considerably in establishing IR spectroscopy as a routine laboratory
characterisation technique, but none more so than the development of commercial
Fourier transform IR (FtIR) spectrometers in the 1960s and 1970s [4, 5] and FtIR
microscopes in the late 1980s [6].
IR spectroscopy is now a routine technique for materials characterisation and has
found numerous applications in forensics [7], environmental science [8] and pharmacology [9]. Raman spectroscopy is a complementary technique with origin in the
discovery of the Raman effect in 1928 [10], for which C.v. Raman was awarded the
Nobel prize in physics in 1930. In 1998 the Raman Effect was designated an ACS
National historic Chemical Landmark, in recognition of its importance in materials
and process analysis. Raman spectroscopy remained largely a curiosity until the advent of the laser in the 1960s, and the revolution in Charged Coupled detector (CCd)
arrays in the 1980s and 1990’s added to the benefits of high laser source intensities.
In addition, the development of narrow band laser line rejection filters meant that the
huge losses in signal from traditional triple monochromator systems could be overcome with the combination of a filter set and a single spectroscopic grating. Furthermore, the significant reductions in acquisition time with multichannel signal detection enabled significant improvements in signal to noise ratios [11]. the combination
of technology developments led to a new range of Raman spectroscopic microscopes
in the 1990s, establishing Raman spectroscopic microscopy as a relatively inexpensive benchtop laboratory tool to rival conventional infrared spectroscopy.
Similar to IR spectroscopy, Raman spectroscopy entails the coupling of incident
radiation with molecular vibrations and the resultant Raman spectrum is characteristic of the compound or material. however, whereas IR spectroscopy involves the
absorption of radiation, Raman spectroscopy is a scattering technique, whereby the
incident radiation couples with the vibrating polarisation of the molecule and thus
generates or annihilates a vibration. the differing underlying mechanisms gives
rise to a complementarity of the two techniques. For a vibration to be active in IR
spectroscopy, a change in dipole is required, whereas to be Raman active, a change
in polarisability is required. vibrations of asymmetric, polar bonds thus tend to be
strong in IR spectra, whereas Raman is particularly suitable as a probe of symmetric, nonpolar groups. Notably, oh vibrations of water are very strong in IR spectra,
whereas they are extremely weak in Raman spectra, rendering Raman a potentially
more suitable technique for bioapplications.
A further implication of the differing physical origins of the techniques is that
whereas IR monitors the absorption of IR radiation, Raman scattering can be employed in the uv, visible or near IR regions of the spectrum. Raman scattering thus offers intrinsically higher spatial resolution for mapping or profiling, the diffraction limit
being determined by the wavelength (< 1 µm for Raman, ~ 5–10 µm for IR). For many
applications, however, near IR is favoured as a source for Raman analysis, to minimise
interference from scattering, fluorescence, or photodegradation of the sample [12].
FtIR has become an accepted tool in biophysics for analysis of the structure
and interactions of proteins [13], lipids [14], carbohydrates [15] and nucleic acids