1. CIRCULAR DICHROISM
Shantan Reddy
VIT University

2. What is it?
• Circular dichroism is the difference in the
absorption of left-handed circularly polarised
light (L-CPL) and right-handed circularly
polarised light (R-CPL).
• Occurs when a molecule contains one or more
chiral chromophores.
Circular dichroism = ΔA(λ) = A(λ)LCPL - A(λ)RCPL
(where λ is the wavelength)

3. Basics of Polarization
Linearly
polarised light Circularly
polarised light

4. Linearly Polarised Light
In a linearly polarised light oscillations are
confined to a single plane. All polarised light
states can be described as a sum of two linearly
polarised states at right angles to each other –
vertically and horizontally polarised light.

5. Vertically polarised
light
Horizontally polarised
light

6. Superposition of the plane polarised
waves

7. Linearly polarised light
Horizontally and vertically polarised light waves
of equal amplitude that are in phase with each
other will produce a resultant light wave which
is linearly polarised at 45˚ and the properties of
the resulting electromagnetic wave depends on
the intensities and phase difference of the
component.
1. Linearly Polarised light

8. Circularly polarised light
• When one of the polarised states is out of phase
with the other by a quarter-wave, the resultant
will be a helix and is known as circularly polarised
light (CPL).
• The optical element that converts between
linearly polarised light and circularly polarised
light is termed a quarter-wave plate. A quarter-
will convert linearly polarised light into circularly
polarised light by slowing one of the linear
components of the beam with respect to the
other so that they are one quarter-wave out of
phase. This will produce a beam of either left- or
right-CPL.

9. 2. Left Circularly Polarised light
3. Right Circularly Polarised light

10. Superposition of circularly polarised
waves
The superposition of a left circularly polarized
wave and a right circularly polarized wave of
equal amplitudes and wavelengths is a plane
polarised wave.

11. Circular Dichroism
• Some materials possess a special
property: they absorb left circularly polarized
light to a different extent than right circularly
polarized light. This phenomenon is
called circular dichroism.
• Assume that a plane-polarized light wave
(blue) traverses a medium that does not
absorb the left circularly polarized component
(red) of the wave at all but highly absorbs the
right circularly polarized component (green).

12. The intensity of the green component decreases in
comparison to the red one.
The superposition of the two components yields a
resulting field vector that rotates along an ellipsoid
path and is called an elliptically polarized light.

13. • The direction of rotation of the elliptically
polarized light is determined by the circular
component that remains stronger after
traversing the material.
• Real materials usually absorb both
components, to a different extent.
• How elliptical the plane-polarized wave
becomes after traversing the medium is
determined by the difference between the
absorptions of the two circularly polarized
components.

14. Circular Birefringence
• There are materials having another special
property: their refraction index is different for left
and right circularly polarized light. This
phenomenon is called circular birefringence.
• Assume that a plane-polarized light wave (blue)
traverses a medium that does not slow down the
left circularly polarized component (red) of the
wave at all but slows down the right circularly
polarized component (green) somewhat. For the
latter component, the refraction index of the
material is n=1.05.

15. This slowdown and the decreased wavelength
is hard to see in the figure because the
refraction index (1.05) is close to 1.0.

16. • Before the medium, the field vectors of the
components coincide when they point vertically
up or down. But after the light exits the
medium, the superposition of the two circularly
polarized components is a plane-polarized wave
with a plane of polarization that is rotated by 36°
with respect to the original polarization plane.
• Real materials usually have refraction indices
greater than 1.0 (but not equal) for both circular
components.
• The angle by which the polarization plane of the
light exiting the medium rotates with respect to
the original polarization plane is determined by
the difference between the refraction indices for
the two circularly polarized components.

17. Circular Dichroism & Circular
Birefringence
The red component traverses the medium
unchanged, but the medium has absorption and a
refraction index with respect to the green component.

18. • The exiting light is no longer plane-polarized, it is
not the plane of polarization that gets rotated but
the big axis of the ellipse of polarization of the
elliptically polarized light.
• With the appropriate instrument, the ellipticity
and the angle of rotation of the polarization
plane of light can be measured. From those
data, the difference between absorptions and
refraction indices with respect to left and right
circularly polarized lights can be calculated.
• Circular dichroism and circular birefringence are
caused by the asymmetry of the molecular
structure of matter. The optical activity of
solutions of biological macromolecules provides
information about the structural properties of the
macromolecules.

19. Physics of CD spectroscopy

20. • In an optically active sample with a different
absorbance ‘A’, for the two components the
amplitude of the stronger absorbed
component will be smaller than the less
absorbed component.
• A projection of the resulting amplitude yields
an ellipse.
• Rotation of the polarization plane by a small
angle ‘a’ occurs when the phases for the two
circular components becomes different which
requires a difference in the refractive index n
by the effect called circular birefringence.

21. • The change of optical rotation with
wavelength is called optical rotatory
dispersion.
• CD and Optical rotation exist together and
they are related by Kronig-Krames
transformation.
• The difference between left and right handed
absorbance is very small ( in the range of
0.0001)
• CD is a function of wavelength making it a
characteristic of molecules.

22. Data Analysis

24. Instrumentation
Prism

25. Nitrogen Purging?
• Removes oxygen from the lamp
housing, monochromator and the sample
chamber.
• The presence of oxygen is detrimental for 2
reasons –
i) when deep UV light strike oxygen, ozone is
produced, which degrades optics.
ii) oxygen absorbs deep UV light which reduces
the amount available for measurement.

26. Sample Preparation and
Measurements
• Additives, Buffers and Stabilizing compounds -
any compound that absorbs in the region of
interest (250 -190 nm) should be avoided.
• Protein solution – It should contain only those
chemicals required to maintain protein
stability at the lowest conc possible. Any
additional protein or peptide will contribute to
the CD signal.
• Data collection – Initial experiments are
needed to establish the parameters for the
actual experiment.

27. Typical Initial Conditions
• Protein Concentration – 0.5mg/ml
• Cell path length – 0.5 mm
• Stabilizers (metal ions, etc.) – minimum
• Buffer concentration – 5mM or as low as
possible while maintaining protein stability.

28. Sample concentration effects
• Optimum absorbance to use is 0.89nm
• For a 1mm path length cell, this absorbance is
achieved with a protein concentration of 0.1-
0.3 mg/ml.

29. CD of proteins and polypeptides
For proteins we will be mainly dealing with the
absorption in the UV region of the spectrum
originated from such chromophores as peptide
bond, amino acid side chains (aromatic side
chains of Phe, Tyr, and Trp have absorption
bands in the vicinity of 250-320 nm; the
disulfide group is an inherently asymmetric
chromophore that can lead to a broad CD
absorption around 250 nm), and any prosthetic
groups.

30. CD of Proteins – Far UV region
n -> π* centered around 220 nm
π -> π* centered around 190 nm
n -> π* involves non-bonding
electrons of O of the carbonyl
π -> π* involves the π-electrons
of the carbonyl
The intensity and energy of
these transitions depends on φ
and ψ (i.e., secondary structure)

31. Far UV-CD of random coil:
positive at 212 nm (π->π*)
negative at 195 nm (n->π*)
Far UV-CD of β-sheet:
negative at 218 nm (π->π*)
positive at 196 nm (n->π*)
Far UV-CD of α-helix:
exiton coupling of the π->π*
transitions leads to positive (π-
>π*)perpendicular at 192 nm
and negative (π->π*)parallel at
209 nm negative at 222 nm is red
shifted (n->π*)

32. Far UV CD spectra and Secondary
Structure of Proteins
After baseline subtraction we
are ready to analyze the data.
Each of the three basic
secondary structures of a
polypeptide chain
(helix, sheet, coil) show a
characteristic CD spectrum. A
protein consisting of these
elements should therefore
display a spectrum that can
be deconvoluted into the
individual contributions.

33. CD of Proteins – Near UV region
PHENYLALANINES TYROSINES TRYPTOPHANS S-S BONDS
Small extinction Lower symmetry and Has the most intense It has a very broad
coefficient due to hence intense absorption band. band.
high symmetry. absorption bands.
Absorption maxima Absorption maxima Absorption maxima is Absorption maxima
at 254, 256, 262 and at 276 nm; hydrogen 282 nm ranges from 250 – 300
267 nm. bonding to the –OH nm
group leads to a red
shift of up to 4nm

34. CD in biochemistry
Used in the understanding of the higher order
structures of chiral macromolecules such as
proteins and DNA. The reason for this is that the CD
spectrum of a protein or DNA molecule is not a sum
of the CD spectra of the individual residues or
bases, but is greatly influenced by the
3-dimension structure of the macromolecule itself.
Each structure has a specific circular dichroism
signature, and this can be used to identify structural
elements and to follow changes in the structure of
chiral macromolecules.

35. The most widely studied circular dichroism
signatures are the various secondary structural
elements of proteins such as the α-helix and the β
sheet. This is understood to the point that CD
spectra in the far-UV (below 260nm) can be used to
predict the percentages of each secondary
structural element in the structure of a protein.

36. There are many algorithms designed for fitting
the circular dichroism spectra of proteins to
provide estimates of secondary structure. The
protein secondary structure CD analysis
software distributed with the Chirascan is CDNN.

37. Other Applications
• CD is a particularly powerful tool to follow
dynamic changes in protein structure which
may result due to the effect of changing
temperature, pH, ligands, or denaturants etc.
• Circular dichroism can be used to follow the
kinetics of refolding of the secondary
structure of a protein using changes in
denaturant concentration.
• It can also be used to follow the unfolding of
proteins by thermal denaturation.

38. • A powerful application of circular dichroism is to
compare two macromolecules, or the same
molecule under different conditions, and
determine if they have a similar structure. This
can be used simply to ascertain if -
i) a newly purified protein is correctly folded,
ii) determine if a mutant protein has folded
correctly in comparison to the wild-type, or
iii) for the analysis of biopharmaceutical products
to confirm that they are still in a correctly folded
active conformation.