Molecular spectroscopy and structure: Optical rotation dispersion and circular dichroism spectra

Question 1

Explain light as a wave

Answer 1

• 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

Question 2

What are polarisers?

Answer 2

• 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

Question 3

What does it mean if a chemical species is ‘optically active’?

Answer 3

when plane polarised light passes through a sample containing such molecules, the plane of polarisation is rotated

Question 4

What is the optical activity derived from?

Answer 4

from the presence of chiral centres in the molecule

Question 5

What property must the source have?

Answer 5

it must be monochromatic (one colour, one wavelength) because the rotation depends on wavelength

Question 6

What is the angle recorded, α?

Answer 6

the angle of rotation of the analyser that is required in order for the plane polarised light to be visible at the eye-piece

Question 7

How is the specific rotation calculated?

Answer 7

[α] = α/lc

where c is the concentration of the optically active species

Question 8

Which units are used for concentration?

Answer 8

grams per 100 cm^3

Question 9

What does dextrorotatory mean?

Answer 9

the medium causes the plane of polarisation to rotate to the right (+)

Question 10

What does levorotatory mean?

Answer 10

the medium causes the plane of polarisation to rotate to the left (-)

Question 11

Why does the plane of polarisation rotate?

Answer 11

because in an optically active medium, the refractive index, n(λ), is different for left and right circularly polarised light

Question 12

How is the angle of rotation determined with respect to the refractive index?

Answer 12

α = π/λ (n(L)-n(R)) l

Question 13

How is the angle of rotation determined in radians?

Answer 13

α = 180/λ (n(L)-n(R)) l

Question 14

What does a spectropolarimeter measure?

Answer 14

how the optical rotation varies with wavelength, giving an optical rotatory dispersion curve (ORD)

Question 15

How is an ORD curve quantified?

Answer 15

by the molar rotation [Φ] = M [α] /100

where M is the molar mass in g.mol^-1

Question 16

What is an interesting property of the optical rotatory dispersion?

Answer 16

It changes sign as the wavelength is scanned through an absorption band

Question 17

What is the Cotton effect?

Answer 17

• 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

Question 18

At which wavelength does the main Cotton effect occur for proteins and polypeptides?

Answer 18

around 200 nm due to absorption by the peptide bond

Question 19

At which wavelength does the Cotton effect occur for nucleic acids?

Answer 19

around 250 to 275 nm due to n—>π* and π—>π*

Question 20

What would happen if the plane of polarisation was to be spun as the light wave propagates?

Answer 20

the magnitude of the electric and magnetic field vectors would remain constant, but their orientation would trace out a pair of helices

Question 21

How can the left and right components of plane polarised light be separated?

Answer 21

using a Fresnel prism

Question 22

What happens if left and right components are subsequently combined?

Answer 22

they give back the plane polarised light

Question 23

What is a circular dichroism spectrum?

Answer 23

a plot of the difference in left and right molar absorptivities against wavelength

Question 24

What does the Cotton effect in ORD correspond to?

Answer 24

to absorption and CD peaks

Question 25

Why have polarimetry measurements of ORD spectra been largely suppplanted by CD?

Answer 25

CD has greater resolving power

Question 26

When can ORD be used?

Answer 26

when the absorption is outside the range of CD instrumentation or obscured by solvent absorption

Question 27

Why do DNA and RNA give significant CD spectra?

Answer 27

although DNA bases are not intrinsically chiral, the double helix provides a chiral environment

Question 28

What is the primary contributer to CD in proteins?

Answer 28

the peptide bond, directly bonded to the chiral Cα

Question 29

What are the differences between CD and ORD and XRD?

Answer 29

• 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

Question 30

Between which wavelengths can common secondary structural types be analysed?

Answer 30

between 260 nm and 180 nm

Question 31

What is the common choice for the standard for comparison with empirical observations?

Answer 31

alpha-helix, beta-sheet and random coil of poly-L-lysine

Question 32

How is the standard used for comparison?

Answer 32

• 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

Question 33

What are the applications of ORD and CD for proteins and polypeptides?

Answer 33

• 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

Question 34

What are the applications of ORD and CD for polynucleotides and nucleic acids?

Answer 34

• evidence for base stacking

- confirmation of the structure of RNA in ribosomes