5 - Plane Polarised Light and CD/ORD Spectroscopy Flashcards
What are plane polarizers made from?
Nicol Prisms are made from rhombohedral calcite (CaCO3) crystals that have been separated and re-attached.
What is the general criterion for optical activity?
Having a non-superimposable mirror image.
How does a polarimeter work?
Light source is passed through a fixed polariser, which enters the polarimeter tube containing the sample. This exits and a second rotatable polarised can be adjusted to find the maximum intensity passing through and hence measuring the angle of rotation.
If the light is rotated clockwise from the viewer’s perspective it is….
Dextrorotatory, (+)
If the light is rotated anti-clockwise from the viewer’s perspective it is….
Levorotatory, (-)
What is specific rotation?
The inherent rotational property of a molecule that allows for comparison without factors such ass concentration and path length.
What two factors must be controlled for a specific rotation value?
Wavelength and temperature.
Why does the plane of polarisation rotate in an optically active medium?
Because of the difference in refractive index between left and right circularly polarised light between the enantiomers.
What does a spectropolarimeter measure?
How the optical rotation of a molecule varies with the wavelength of light used.
What is the graphical result of a spectropolarimeter?
An Optical Rotatory Dispersion curve (ORD)
How is the optical rotation of molecules often quantified for use in spectropolarimetry?
Molar rotation: (molar mass x specific activity)/100
How is the optical rotation of macromoleculesmolecules often quantified for use in spectropolarimetry?
Per mole of repeating unit or per mean residue molar mass.
What is the refractive index?
Speed of light in vacuum/speed of light through given medium.
What is the Cotton Effect?
The change in sign of the optical rotation on an ORD as the wavelength scans through an absorption band.
What are the main wavelengths at which the Cotton Effect occurs for polypeptides and nucleic acids?
Peptide bond - 200nm
Nucleotide bases - 250-270nm
What is used to separate left and right circularly polarised light?
A Fresnel Prism.
How do chiral species interact with circularly polarise light?
A chiral molecule will absorb left and right circularly polarised light to a different extent, so will have different extinction coefficients for the two polarisations.
What is a circular dichroism spectrum a plot of?
Difference in molar absorptivity of a chiral molecule between left and right circularly polarised light (y) against wavelength (x).
EL - ER vs. lambda
How is the Cotton effect visulalised on a CD spectrum?
It is the at peaks of the difference in extinction coefficient.
Is CD or ORD more widespread, and why?
CD as it has greater resolving power.
When is ORD more likely to be used?
When the absorption is out of range for CD or is obscured by solvent absorption.
Why do DNA and RNA give significant CD spectra?
The double helix is an intrinsically chiral environment, and purines and pyrimidines both absorb inherently.
What wavelength do DNA and RNA show significant CD spectrum character?
Near and far UV regions.
Why is CD spectroscopy able to be used on proteins?
The peptide bond is the primary contributor, and its absorption will differ depending on the environment - the resulting spectrum being an average of all the configurations present.
How does the structural information from CD and ORD spectra compare to X-ray diffraction results?
Absolute structural information far less detailed, but is useful for comparison/conformation - particularly as unlike XRD CD and ORD can be done in solution and hence more likely native state.
Why is CD useful for dynamic studies and what does this make it useful for?
It is highly sensitive to changes in conformation, so is used to study/monitor binding events and folding/unfolding.
What level of order is CD especially sensitive to?
Secondary structure, analysis of a-helix and parallel/antiparallel B-sheet/turn etc the most prominent use.
What wavelengths are used in CD for secondary structure analysis?
180 and 260nm
What standard is often used in order to weight the helical/other character of a protein in CD spectroscopy?
Poly-L-Lysine
In what fashion do the contributions to the CD spectra of different secondary structures combine?
Linearly, or so it is assumed.
What applications does CD have for nucleic acid study?
RNA conformation in structures such as ribosomes.
Providing evidence for base-stacking.
