65
4 Raman optical Activity of Biological Samples
viruses. RoA data below ~ 600 cm
−1
are often unreliable from highly scattering
samples due to offsets associate with the intense Rayleigh wing.
Although ICP RoA instruments of the type described above have established the
value of RoA and will continue to be useful, a completely new design of RoA instrument with significant advantages inherent to the SCP strategy has recently been
developed by hug [19, 25]. In particular, ‘flicker noise’ arising from dust particles
and density fluctuations in the sample, laser power fluctuations, etc. are eliminated
as the intensity difference measurements required to extract the circularly polarized
components of the scattered beam are taken between two orthogonal components
of the scattered light measured simultaneously over the same acquisition period.
this strategy cancels out the flicker noise, resulting in greatly superior signal-tonoise characteristics. the basic design is illustrated in Fig. 4.3 which corresponds to
hug’s original implementation of the SCP strategy [25]; some of the details differ in
later versions, but the basic principle is the same. the incident laser beam, the initial
linear polarization state of which is ‘scrambled’ by a fast rotation of its azimuth, is
deflected by a very small right-angle prism into the sample cell. the cone of backscattered light is collimated onto a liquid crystal retarder set to convert right- and
left-circular polarization states into linear polarization states with azimuths perpendicular and parallel, respectively, to the plane of the instrument. A beam-splitting
cube then diverts the perpendicular component at 90
o
to the propagation direction
of the parallel component, which passes through undiverted. In this way, the rightand left-circularly polarized components of the backscattered light are separated
and collected into the ends of two fiber optic cables. Each fiber optic converts the
cross section from circular at the input end to a linear configuration at the output
end that matches the entrance slit of the spectrograph, thereby enabling separate Raman spectra for the right- and left-circularly polarized components of the scattered
light to be dispersed simultaneously, one above the other, on the CCd. the ICP
RoA spectrum then corresponds to a difference in the small circularly polarized
components in the Raman bands for a chiral molecule. Small differences in the two
detection channels are compensated by interconverting their function through the
switching of the liquid crystal retarder from the − λ/4 to the + λ/4 state. A commercial instrument based on this new design that also incorporates a sophisticated artefact suppression protocol, based on a ‘virtual enantiomers’ approach which greatly
facilitates the routine acquisition of reliable RoA spectra [26], is now available (the
ChiralRAMAN spectrometer from Biotools, Inc.).
4.3 Applications of Computational Approach to Analysis
of ROA Spectra of Biomolecules
the first RoA spectra were published 40 years ago [2] and from that time molecular
structures of hundreds of biomolecules were investigated in their native environments by vibrational Raman optical activity. Carbohydrates that play an essential
role in various biological processes are one of many excellent examples that show
the power of the RoA technique. they give prominent RoA signals over a wide
4 Raman optical Activity of Biological Samples
viruses. RoA data below ~ 600 cm
−1
are often unreliable from highly scattering
samples due to offsets associate with the intense Rayleigh wing.
Although ICP RoA instruments of the type described above have established the
value of RoA and will continue to be useful, a completely new design of RoA instrument with significant advantages inherent to the SCP strategy has recently been
developed by hug [19, 25]. In particular, ‘flicker noise’ arising from dust particles
and density fluctuations in the sample, laser power fluctuations, etc. are eliminated
as the intensity difference measurements required to extract the circularly polarized
components of the scattered beam are taken between two orthogonal components
of the scattered light measured simultaneously over the same acquisition period.
this strategy cancels out the flicker noise, resulting in greatly superior signal-tonoise characteristics. the basic design is illustrated in Fig. 4.3 which corresponds to
hug’s original implementation of the SCP strategy [25]; some of the details differ in
later versions, but the basic principle is the same. the incident laser beam, the initial
linear polarization state of which is ‘scrambled’ by a fast rotation of its azimuth, is
deflected by a very small right-angle prism into the sample cell. the cone of backscattered light is collimated onto a liquid crystal retarder set to convert right- and
left-circular polarization states into linear polarization states with azimuths perpendicular and parallel, respectively, to the plane of the instrument. A beam-splitting
cube then diverts the perpendicular component at 90
o
to the propagation direction
of the parallel component, which passes through undiverted. In this way, the rightand left-circularly polarized components of the backscattered light are separated
and collected into the ends of two fiber optic cables. Each fiber optic converts the
cross section from circular at the input end to a linear configuration at the output
end that matches the entrance slit of the spectrograph, thereby enabling separate Raman spectra for the right- and left-circularly polarized components of the scattered
light to be dispersed simultaneously, one above the other, on the CCd. the ICP
RoA spectrum then corresponds to a difference in the small circularly polarized
components in the Raman bands for a chiral molecule. Small differences in the two
detection channels are compensated by interconverting their function through the
switching of the liquid crystal retarder from the − λ/4 to the + λ/4 state. A commercial instrument based on this new design that also incorporates a sophisticated artefact suppression protocol, based on a ‘virtual enantiomers’ approach which greatly
facilitates the routine acquisition of reliable RoA spectra [26], is now available (the
ChiralRAMAN spectrometer from Biotools, Inc.).
4.3 Applications of Computational Approach to Analysis
of ROA Spectra of Biomolecules
the first RoA spectra were published 40 years ago [2] and from that time molecular
structures of hundreds of biomolecules were investigated in their native environments by vibrational Raman optical activity. Carbohydrates that play an essential
role in various biological processes are one of many excellent examples that show
the power of the RoA technique. they give prominent RoA signals over a wide