5 - Plane Polarised Light and CD/ORD Spectroscopy

Question 1

What are plane polarizers made from?

Answer 1

Nicol Prisms are made from rhombohedral calcite (CaCO3) crystals that have been separated and re-attached.

Question 2

What is the general criterion for optical activity?

Answer 2

Having a non-superimposable mirror image.

Question 3

How does a polarimeter work?

Answer 3

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.

Question 4

If the light is rotated clockwise from the viewer’s perspective it is….

Answer 4

Dextrorotatory, (+)

Question 5

If the light is rotated anti-clockwise from the viewer’s perspective it is….

Answer 5

Levorotatory, (-)

Question 6

What is specific rotation?

Answer 6

The inherent rotational property of a molecule that allows for comparison without factors such ass concentration and path length.

Question 7

What two factors must be controlled for a specific rotation value?

Answer 7

Wavelength and temperature.

Question 8

Why does the plane of polarisation rotate in an optically active medium?

Answer 8

Because of the difference in refractive index between left and right circularly polarised light between the enantiomers.

Question 9

What does a spectropolarimeter measure?

Answer 9

How the optical rotation of a molecule varies with the wavelength of light used.

Question 10

What is the graphical result of a spectropolarimeter?

Answer 10

An Optical Rotatory Dispersion curve (ORD)

Question 11

How is the optical rotation of molecules often quantified for use in spectropolarimetry?

Answer 11

Molar rotation: (molar mass x specific activity)/100

Question 12

How is the optical rotation of macromoleculesmolecules often quantified for use in spectropolarimetry?

Answer 12

Per mole of repeating unit or per mean residue molar mass.

Question 13

What is the refractive index?

Answer 13

Speed of light in vacuum/speed of light through given medium.

Question 14

What is the Cotton Effect?

Answer 14

The change in sign of the optical rotation on an ORD as the wavelength scans through an absorption band.

Question 15

What are the main wavelengths at which the Cotton Effect occurs for polypeptides and nucleic acids?

Answer 15

Peptide bond - 200nm

Nucleotide bases - 250-270nm

Question 16

What is used to separate left and right circularly polarised light?

Answer 16

A Fresnel Prism.

Question 17

How do chiral species interact with circularly polarise light?

Answer 17

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.

Question 18

What is a circular dichroism spectrum a plot of?

Answer 18

Difference in molar absorptivity of a chiral molecule between left and right circularly polarised light (y) against wavelength (x).

EL - ER vs. lambda

Question 19

How is the Cotton effect visulalised on a CD spectrum?

Answer 19

It is the at peaks of the difference in extinction coefficient.

Question 20

Is CD or ORD more widespread, and why?

Answer 20

CD as it has greater resolving power.

Question 21

When is ORD more likely to be used?

Answer 21

When the absorption is out of range for CD or is obscured by solvent absorption.

Question 22

Why do DNA and RNA give significant CD spectra?

Answer 22

The double helix is an intrinsically chiral environment, and purines and pyrimidines both absorb inherently.

Question 23

What wavelength do DNA and RNA show significant CD spectrum character?

Answer 23

Near and far UV regions.

Question 24

Why is CD spectroscopy able to be used on proteins?

Answer 24

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.

Question 25

How does the structural information from CD and ORD spectra compare to X-ray diffraction results?

Answer 25

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.

Question 26

Why is CD useful for dynamic studies and what does this make it useful for?

Answer 26

It is highly sensitive to changes in conformation, so is used to study/monitor binding events and folding/unfolding.

Question 27

What level of order is CD especially sensitive to?

Answer 27

Secondary structure, analysis of a-helix and parallel/antiparallel B-sheet/turn etc the most prominent use.

Question 28

What wavelengths are used in CD for secondary structure analysis?

Answer 28

180 and 260nm

Question 29

What standard is often used in order to weight the helical/other character of a protein in CD spectroscopy?

Answer 29

Poly-L-Lysine

Question 30

In what fashion do the contributions to the CD spectra of different secondary structures combine?

Answer 30

Linearly, or so it is assumed.

Question 31

What applications does CD have for nucleic acid study?

Answer 31

RNA conformation in structures such as ribosomes.

Providing evidence for base-stacking.