Optical rotatory dispersion is the variation in the optical rotation of a substance with a change in the wavelength of light. For wavelengths that are absorbed by the optically active sample, the two circularly polarized components will be absorbed to differing extents. This unequal absorption is known as circular dichroism.

1. Optical Rotary Dispersion &
Circular Dichriosm spectroscopy
By-Lovnish Thakur
ASU2014010100099
3rd SEM, Integrated Biotech

2. Optical rotatory dispersion & Circular
Dichroism
 Optical rotatory dispersion is the variation in
the optical rotation of a substance with a
change in the wavelength of light.
 For wavelengths that are absorbed by the
optically active sample, the two circularly
polarized components will be absorbed to
differing extents. This unequal absorption is
known as circular dichroism.

3. Polarized Light
• Light with
oscillations
confined to a single
plane.
• Top: vertically
polarized light
• Bottom:
horizontally
polarized light
http://www.photophysics.com/polarization.php

4. Circularly Polarized Light
Sum of vertically and horizontally plane
polarized light in which the phases differ by a
quarter wave
http://www.photophysics.com/polarization.php

5. Circular Dichroism (CD) Spectroscopy
• Difference in absorption between left-handed
and right-handed circularly polarized light as a
function of wavelength
• Unlike optical rotatory dispersion (ORD)
spectroscopy, which only requires a chiral
molecule, CD requires a chromophore in a
chiral environment.

6. ORD – why isn’t a chromophore required?
All chiral molecules exhibit circular birefringence, basically the
rates of propagation of left and right-handed polarized light
(which can be added to form plane-polarized light) are
different. This changes their relative phase, and therefore the
observed angle at the detector.
http://www.photophysics.com/polarization.php

7. Origin of Ellipticity Unit
http://www.photophysics.com/polarization.php

8. Chromophores in Biomolecules
• Proteins
– Aromatic sidechains (π-π* ~280 nm, near UV)
• Phenylalanine (ε ~ 250 M-1cm-1)
• Tyrosine (ε ~ 1000 M-1cm-1)
• Tryptophan (ε ~ 5000 M-1cm-1)
– Backbone amide bond (far UV)
• n-π* ~210-220 nm (major peak in UV spectrum, (ε ~
100 M-1cm-1)
• π-π* ~190 nm
Sensitive to tertiary
structure
Sensitive to
secondary structure

9. Working
Measurements carried out in the visible and
ultra‐violet region of the electro‐magnetic
spectrum monitor electronic transitions, and,
if the molecule under study contains chiral
chromophores then one CPL state will be
absorbed to a greater extent than the other
and the CD signal over the corresponding
wavelengths will be non‐zero.

10. • There is a source of monochromatic linearly polarised light
which can be turned into either left‐ or right‐circularly
polarised light
• A circular dichroism spectrophotometer has a specialised
optical element called a photo‐elastic modulator (PEM),
which produce left‐ and then right‐ circularly polarised light at
the drive frequency.
• On the other side of the sample position there is a light
detector.
• When there is no circularly dichroic sample in the light path,
the light hitting the detector is constant.
• If there is a circularly dichroic sample in the light path, the
recorded light intensity will be different for right‐ and left‐CPL.

11. thz.yale.edu

12. Applications
• A large subset of the use of circular dichroism in biochemistry is in the
understanding of the higher order structures of chiral
macromolecules such as proteins and DNA
• Mostly it is widely used to study various secondary structural
elements of proteins such as the α‐helix and the β sheet because CD
spectra in the far‐UV (below 260 nm) can be used to predict the
percentages of each secondary structural element in the structure of a
protein.
• Circular dichroism is to compare two macromolecules, or the same
molecule under different conditions, and determine if they have a
similar structure.
• For the analysis of biopharmaceutical products to confirm that they
are still in a correctly folded active conformation.

13. • In this they examined the thermal denaturation of ribonuclease T1 by CD
using both the original and expanded sets of reference proteins and
obtained more consistent results with the expanded set. The expanded set
of reference proteins will be helpful for the determination of protein
secondary structure from protein CD spectra with higher reliability,
especially of proteins with significant unordered structure content and/or in
the course of denaturation.
• The basic principle involved in the analysis of protein CD spectra, and used
in the estimation of secondary structure fractions, is that the protein CD
spectrum can be expressed as a linear combination of CD spectra of
individual secondary structure components. Spectra of model polypeptides
or of a set of reference proteins with known structures are used to
determine the component secondary structure spectra.

14. Conclusion
Circular Dichriosm spectroscopy is a very useful
technique to study the structure of
biomolecule(chiral molecule). And it is most
widely used to study secondary structure of
proteins.

15. References
• http://naldc.nal.usda.gov/download/31237/P
DF
• http://www.chem.uci.edu/~dmitryf/manuals/
Fundamentals/CD%20spectroscopy.pdf
• http://www.uic.edu/orgs/ctrstbio/manuals/ad
ler