Molecular spectroscopy and structure: Optical rotation dispersion and circular dichroism spectra Flashcards
Explain light as a wave
- two components: an electric field component and a magnetic field component
- propagate at the same speed, in the same direction, in phase but at right angles to one another
- normal (unpolarised) light is comprised of waves with the electric field pointing in all possible directions
What are polarisers?
- polarisers are usually Nicol prisms
- these are rhombohedral crystals of calcite (CaCO3) that are essentially cut and glued back together
- the refraction that takes place at the join between the two parts of the prism is what gives rise to the plane polarisation
What does it mean if a chemical species is ‘optically active’?
when plane polarised light passes through a sample containing such molecules, the plane of polarisation is rotated
What is the optical activity derived from?
from the presence of chiral centres in the molecule
What property must the source have?
it must be monochromatic (one colour, one wavelength) because the rotation depends on wavelength
What is the angle recorded, α?
the angle of rotation of the analyser that is required in order for the plane polarised light to be visible at the eye-piece
How is the specific rotation calculated?
[α] = α/lc
where c is the concentration of the optically active species
Which units are used for concentration?
grams per 100 cm^3
What does dextrorotatory mean?
the medium causes the plane of polarisation to rotate to the right (+)
What does levorotatory mean?
the medium causes the plane of polarisation to rotate to the left (-)
Why does the plane of polarisation rotate?
because in an optically active medium, the refractive index, n(λ), is different for left and right circularly polarised light
How is the angle of rotation determined with respect to the refractive index?
α = π/λ (n(L)-n(R)) l
How is the angle of rotation determined in radians?
α = 180/λ (n(L)-n(R)) l
What does a spectropolarimeter measure?
how the optical rotation varies with wavelength, giving an optical rotatory dispersion curve (ORD)
How is an ORD curve quantified?
by the molar rotation [Φ] = M [α] /100
where M is the molar mass in g.mol^-1
What is an interesting property of the optical rotatory dispersion?
It changes sign as the wavelength is scanned through an absorption band
What is the Cotton effect?
- the characteristic change in optical rotatory dispersion and/or/ circular dichroism
- if the wavelength is decreased, the rotation angle increases until it reaches a maximum and then decreases, passing through zero at the wavelength at which the maximum of absorption occurs
- as the wavelength is decreased further, the angle becomes negative, until it reaches a minimum, after which is rises again
At which wavelength does the main Cotton effect occur for proteins and polypeptides?
around 200 nm due to absorption by the peptide bond
At which wavelength does the Cotton effect occur for nucleic acids?
around 250 to 275 nm due to n—>π* and π—>π*
What would happen if the plane of polarisation was to be spun as the light wave propagates?
the magnitude of the electric and magnetic field vectors would remain constant, but their orientation would trace out a pair of helices
How can the left and right components of plane polarised light be separated?
using a Fresnel prism
What happens if left and right components are subsequently combined?
they give back the plane polarised light
What is a circular dichroism spectrum?
a plot of the difference in left and right molar absorptivities against wavelength
What does the Cotton effect in ORD correspond to?
to absorption and CD peaks
Why have polarimetry measurements of ORD spectra been largely suppplanted by CD?
CD has greater resolving power
When can ORD be used?
when the absorption is outside the range of CD instrumentation or obscured by solvent absorption
Why do DNA and RNA give significant CD spectra?
although DNA bases are not intrinsically chiral, the double helix provides a chiral environment
What is the primary contributer to CD in proteins?
the peptide bond, directly bonded to the chiral Cα
What are the differences between CD and ORD and XRD?
- CD and ORD spectra are recorded in solution, whereas XRD takes place with solid samples
- the molecular structure will not always be similar in the two different phases
- the CD spectra have the advantage of great sensitivity to changes in conformation, whereas XRD can only be used to demonstrate absolute conformation
- not all proteins can form crystals
Between which wavelengths can common secondary structural types be analysed?
between 260 nm and 180 nm
What is the common choice for the standard for comparison with empirical observations?
alpha-helix, beta-sheet and random coil of poly-L-lysine
How is the standard used for comparison?
- the measured protein ORD and CD spectra and fitted using a weighted sum of the spectra from the standards
- the weight of the contributions from each of the standards thus reflects the percentage contribution from the corresponding conformation
- it is assumed that the conformations combine in a linear fashion
What are the applications of ORD and CD for proteins and polypeptides?
- enzyme binding to substrate, coenzyme or inhibitor (the magnitude of the change is proportional to the number of binding molecules)
- denaturation of proteins and protein folding
What are the applications of ORD and CD for polynucleotides and nucleic acids?
- evidence for base stacking
- confirmation of the structure of RNA in ribosomes
