The document discusses concepts related to natural light, including its polarized and unpolarized forms, as well as phenomena such as optical activity and circular dichroism (CD). It explains the principles behind various types of polarized light, instrumentation for measurement, and applications of CD in analyzing protein structures based on their chiral nature. Additionally, it covers optical rotation and dispersion effects, including the Cotton effect and their implications in biochemistry.

1. CD & ORD
INSTRUMENTATION, APPLICATION, &
PRINCIPLE

2. Natural LightNatural light is having two
components electric component
and magnetic component, and
both are perpendicular to each
other in different planes.
Both these electric component
and magnetic components are also
perpendicular to the direction of
propagation of light.
NATURAL LIGHT

3. UNPOLARISED LIGHT
POLARISED LIGHT
NATURAL LIGHT
ordinary (non-polarized)
light consists of many
beams vibrating in
different planes
plane-polarized light consists
of only those beams that
vibrate in the same plane

4. OPTICAL ACTIVITY
• The compounds which are having the ability to rotate
the plane of polarized light are called optical active
compound.
• This property of compounds is called optical activity.
• Measured by Polarimeter
• Dextrorotatory - sugars
• Levorotatory – proteins, phospholipids

5. Rotation of plane-polarized light

7. TYPES OF POLARIZED LIGHT
I. Linear or plane polarized light.
II. Circular polarized light.
III.Elliptically polarized light.

8. Linearly
polarised light Circularly
polarised light

9. 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.

10. Vertically polarised light

11. Horizontally polarised light

12. Superposition of the plane polarised waves
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.

13. Linearly Polarised light

14. • 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).
CIRCULARLY POLARISED LIGHT
• The optical element that converts between linearly
polarised light into circularly polarised light is termed a
quarter-wave plate.

15. • A quarter-plate 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.
CIRCULARLY POLARISED LIGHT

16. Left Circularly Polarised light
Anti-clockwise

17. Right Circularly Polarised light
Clockwise

18. 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.

19. • When the absorption at a given frequency is different in different
directions, the molecule will appear to have a different color when
viewed with two kinds of plane polarized light. This phenomenon is
dichroism.
 In the region where the molecules absorb light, there occurs a
preferential absorption of one of the circularly polarized components.
This difference in the absorption rates, which takes place along with
differential retardation due to the circular birefringence, results in
circular dichroism (CD), that is, the optically active medium has an
unequal molar absorption coefficient (є) for left and right circularly
polarised lights.
CIRCULAR DICHROISM

20.  The molar absorption coefficient, molar extinction coefficient,
or molar absorptivity (ε), is a measurement of how strongly a chemical
species absorbs light at a given wavelength.
 It is an intrinsic property of the species; the actual absorbance, A,
of a sample also depends on the path length, ℓ, and the
concentration, c, of the species, according to the Beer-Lamberts law.
Molar absorption coefficient (ε) ?

21. CIRCULAR DICHROISM
Circular dichroism is the difference in molar absorptivity.
∆ є = єL - єR ≠ 0

22. • 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).
CIRCULAR DICHROISM

23. • 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.

24. • 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.
Extent of Ellipticity ??

25. Linear polarized light can be viewed
as a superposition of opposite
circular polarized light of equal
amplitude and phase
different absorption of the left- and
right hand polarized component leads
to ellipticity (CD) and optical rotation
(OR).

26.  The two circularly polarised components, which emerge from the
optically active medium, are out of phase with each other and also are
of unequal amplitude. The vector sum resulted, traces out an elliptical
path instead of vibrating in a single line.
 Thus the linearly polarised light gets transformed to elliptically
polarised light due to the unequal absorption of the two components
within.
 The angle between the major axis of ellipse and the plane of
original radiation is the angle of rotation α. The elliptically θ is the
angle whose tangent is the ratio of the minor axis of ellipse OA to
major axis OB.

27. • CD is measurement of how an optically active compound absorbs
right- and left-handed circularly polarized light.
• All optically active compounds exhibit CD in the region of the
appropriate absorption band.
• CD is plotted as l-r i.e [θ] vs 
• For CD, the resulting transmitted radiation is not plane-polarized but
elliptically polarized.

28.    
l
AA RL
4
303.2
cmrad 1- 

 ellipticity
l path length through the sample
A absorption
Circular dichroism graphs are plots of [θ] against wavelengths.

29. Refraction is the bending of a wave when it enters a medium
where its speed is different. The refraction of light when it
passes from a fast medium to a slow medium bends the light ray
toward the normal to the boundary between the two media.
Refraction ? Refractive index ?

30. Circular Birefringence
Fresnel postulated that when the circularly polarized light beams
pass through an optically active medium, the refractive index for
one of the circularly polarized component will be different from
that for the other, then the medium is said to be circularly
birefringent.
That is
nL -nR ≠ 0

31. • The difference in refractive indices indicates difference in light
velocities.
• The emerging beams will be out of phase with each other and the
resultant vector is rotated by an angle α to the origina plane of
polarization.
• The angle of rotation, α, in degress per decimeter is obtained by
α = 1800 ( nL - nR )
λ (cm)
Circular Birefringence

32. The red component traverses the medium unchanged, but the
medium has absorption and a refraction index with respect to the
green component.

33. Instrumentation
Dicographs, dichrometers or
CD spectrometers

34. Instrumentation

35. • Light source is typically a 450 watt high pressure xenon arc lamp. The light
is dispersed by the monochromator.
• The monochromatic light which is passed through an exit slit gets linearly
polarised in the polariser. The plane-polarised light is then allowed to pass
through the sample cell ( in case of ORD) and through wave plate (in case of
CD).
•The modulated signal is detected by photomultiplier and is fed to an
amplifier and then to the recorder.
Instrumentation

36. • A waveplate or retarder is an optical device that alters the polarization state
of a light wave travelling through it.
•Two common types of wave plates are the half-wave plate, which shifts the
polarization direction of linearly polarized light, and the quarter-wave plate,
which converts linearly polarized light into circularly polarized light and vice
versa.
•Wave plates are constructed out of a birefringent material (such
as quartz or mica), for which the index of refraction is different for different
orientations of light passing through it.
•The behavior of a wave plate depends on the thickness of the crystal, the
wavelength of light, and the variation of the index of refraction.
Wave plate

38. • A wave plate that imparts a retardation of λ/4 (known as a quarter wave
plate) will convert linearly polarized light to circularly polarized light if the
input polarization is aligned at 45° to the optical axis, or vice versa.
•A typical wave plate is simply a birefringent crystal with a carefully chosen
orientation and thickness.
•The crystal is cut into a plate, with the orientation of the cut chosen so that
the optic axis of the crystal is parallel to the surfaces of the plate.
•The solvents used to dissolve the sample for the measurement of ORD/ CD
should not be optically active or should not react with the sample.
• Ethanol, methanol and water are the solvents of choice.

39. • ORD is technique related to the optical activity.
• Rate of change of specific rotation with change in wave length.
• Optical rotation caused by compound changed with wavelength
of light was first noted by Biot in 1817.
OPTICAL ROTATORY DISPERSION

40. • Optical rotation varies with wavelength of the radiation.
• This dependence of optical rotation on wavelength is called
optical rotatory dispersion (ORD).
• An optically active sample gives rise to optical rotation even at
wavelengths which do not fall within region in which sample
absorbs.
OPTICAL ROTATORY DISPERSION

41. • Optical rotation α is given by
α = 180 b/ wavelength ( nL - nR )
• Common form in which optical rotation is reported is either
specific rotation [α] or molar rotation (m’)
[α] = 100 α/ C b where C = concentration (gm/cm3) and b = light
path (decimeter)
M’ = [α] M/ 100
Where M= molecular weight

42. Instrumentation
• ORD is measured by a spectropolarimeter. Basic parts are :
1] light source 2] monochromator c] polarizer 4] analyzer
5] sample tubes 6] photomultiplier
 Light source – tungsten filament lamp
 For specific wavelength – xenon- or mercury vapor lamps are
used.
 Polarizer : Nicol prism , Glan-Thompson prism or polaroid filter

43.  TYPES OF ORD CURVES:
They are of two types
 1) Plain curves
 2) Anamolous curves
a) Single cotton effect curves
b) Multiple cotton effect curves
 The curves obtained do not contain any peak and that curve do
not cross the zero rotation line.
 Such waves are obtained for compounds which do not have
absorption in the wavelength region where optical activity is
being examined.

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M.M.C.P.

45. Anamolous curvesː
 These curve on the other hand shows a number of extreme
peaks and troughs depending on the number of absorbing
groups and therefore known as anomolous dispersion of optical
rotation.
 This type of curve is obtained for compounds ,which contain an
asymmetric carbon atom and also contains chromophore.
Single cotton effect curvesː
 These are anomolous dispersion curves which shows maximum
and minimum both of them occurring in the region of maximum
absorption.
 If an approaching the region of cotton effect from the long
wavelength ,one passes first through maximum (peak) and then a
minimum (trough) ,the cotton effect said to be positive .
(Positive Cotton effect is where the peak is at a higher
wavelength than the trough).

46. • If a plot of rotation against wavelength shows maxima and
minima then it is called as Cotton effect .
 A Cotton effect is positive if the peak occurs at longer
wavelength.
 A Cotton effect is negative is the trough is at shorter
wavelength.
COTTON EFFECT

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M.M.C.P.

48.  If the trough is reached first and the peak it is called a
negative cotton effect curves.
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M.M.C.P.

49. CD is used for proteins because of the chiral nature of the
structural features of proteins - Biopolymers are intrinsically
asymmetric; L-amino acids predominate over D-amino acids.
 Amino acids
Chromophores PHE, TRP, TYR
When an aromatic residue is held rigidly in space, its environment is
asymmetric, and it will exhibit circular dichroism.
 Amide bond
In secondary structure conformations, the backbone and the amide
bond chromophores are arranged in regular, organized, asymmetric
patterns.
APPLICATIONS OF CD

50. Near-UV CD spectroscopy is dominated by Phe, Tyr, Trp and disulfides
Near UV CD (250 - 350 nm)
The amide group is the most abundant CD chromophore in proteins.
* transition ~ 190 nm
n* transition ~220 nm
Far UV CD (180 - 250 nm)

51. Far UV CD
exhibits distinct
spectra for
-helical,
-sheet,
and random coil
secondary structure.

52. 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.

53. 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*)

54.  CD exhibits characteristic spectra for protein secondary structure
features - alpha helix, beta sheet random coil
Primary uses for CD:
• analyze structural changes in a protein upon some perturbation.
• compare the structure of a mutant protein to the parent protein.
• screen candidate proteins for more detailed structural analysis
(NMR or X-ray crystallography).