This document provides an introduction to circular dichroism (CD) spectroscopy, explaining its principles, including the measurement of light absorption differences between left- and right-handed polarized light due to structural asymmetry. It details the types of polarized light, the effects of optical rotation and dispersion, and the relationship between CD and optical properties of optically active compounds. Additionally, it outlines sample preparation techniques and data collection standards to optimize CD measurements.

1. Circular Dichroism
Part I. Introduction

2. Circular Dichroism
Circular dichroism (CD) spectroscopy measures differences
in the absorption of left-handed polarized light versus right-
handed polarized light which arise due to structural asymme
try. The absence of regular structure results in zero CD inte
nsity, while an ordered structure results in a spectrum which
can contain both positive and negative signals.
Jasco J-810 Circular Dichroism System

3. Chiral structure can be distinguished and charact
erized by polarized light
Optical rotation
Optical rotation: the rotation of linearly polarized light by t
he sample
Optical rotary dispersion
Optical rotary dispersion: the variation of optical rotatio
n as a function of wavelength. The spectrum of optical rotation.
Circular Dichroism
Circular Dichroism: the difference in absorption of left an
d right circularly light.

4. Types of polarized light
• Plane polarized light consists two circularly
polarized components of equal intensity
• Two circularly polarized components are like left-
and right-handed springs
• As observed by looking at the source, right-
handed circularly polarized light rotates clockwise
• Frequency of rotation is related to the frequency of
the light
• Can be resolved into its two circularly polarized
components
• When added together after passing through an
optically isotropic medium, plane polarized light
results

6. Polarized Light
Linear Polarized Light
 
  0
,
2
sin
2
sin
)
,
( 0
0











t
z
E
ct
z
E
t
c
z
E
t
z
E
y
x




7. Passing plane polarized light through a bir
efringent plate (in the z-direction) which s
plits the light into two plane-polarized bea
ms oscillating along different axes (e.g., fa
st along x and slow along y). When one of
the beams is retarded by 90º (using a quart
er-wave retarder) then the two beams whic
h are now 90º out of phase are added toget
her, the result is circularly polarized light
of one direction. By inverting the two axes
such that the alternate beam is retarded tha
n circularly polarized light of the other dir
ection is generated.
The result of adding the right and left circu
larly polarized that passes through the opti
cally active sample is elliptically polarized
light, thus circular dichroism is equivalent
to ellipticity
Circular Polarized Light

8. Polarized Light
Circularly Polarized Light
 
  
















4
1
2
sin
,
2
sin
,
0
0






ct
z
E
t
z
E
ct
z
E
t
z
E
y
x  
  

















4
1
2
sin
,
2
sin
,
0
0






ct
z
E
t
z
E
ct
z
E
t
z
E
y
x
Left-handed right-handed

9. Optical rotary dispersion
• If the refractive indices of the sample for the left a
nd right handed polarized light are different, when
the components are recombined, the plane-polariz
ed radiation will be rotated through an angle 
• nl, nr are the indices of the refraction for left-hande
d and right-handed polarized light
  is in radians per unit length (from )
λ
n
n
α r
l 


10. Optical Rotation
   
R
L n
n 





1
-
cm
rad
rotation
n refractive index
wavelength of light
angle of rotation

11. Optical Rotation
• Usually reported as a specific rotation [],
measured at a particular T, concentration and 
(normally 589; the Na D line)
• Molar rotation [] = []MW10-2
 
mL
100
g
c
decimeters
in
pathlength
lc
α
10
α
2



l

12. Optical rotary dispersion
 
d'
c'
α
α 
• Concentration of an optically active substance, c’, expressed in g cm-1
(as density of a pure substance)
• d’ = thickness of the sample in decimeters
   
'
'
10
α
10
α
2
2
d
c
M
M
M

 



• M = molecular weight of the optically active component
• the 10-2
factor is subject to convention and is not always included in
[M]

13. Optical rotary dispersion
   
'
'
10
α
10
α
2
2
d
c
M
M
M

 



• M = molecular weight of the optically
active component
• n. b. the 10-2
factor is subject to convention
and is not always included in [M]

14. Optical rotary dispersion
• ORD curve is a plot of molar rotation [] or [M] vs 
• Clockwise rotation is plotted positively; counterclockw
ise rotation is plotted negatively
• ORD is based solely on the index of refraction
• So-called plain curve is the ORD for a chiral compoun
d that lacks a chromophore
• Chiral compounds containing a chromophore can give
anomalous, or Cotton effect, curves

15. Cotton Effect
•Positive Cotton effect is where t
he peak is at a higher wavelength
than the trough
•Negative Cotton effect is the op
posite
•Optically pure enantiomers alwa
ys display opposite Cotton effect
ORD curves of identical magnitu
de
•Zero crossover point between th
e peak and the trough closely cor
responds to the normal UV max

16. Circular Polarized Light

17. Circular Polarized Light

18. Circular dichroism
• Measurement of how an optically active compound absorb
s right- and left-handed circularly polarized light
• All optically active compounds ex-hibit CD in the region o
f the appropriate absorption band
• CD is plotted as l-r vs 
• For CD, the resulting transmitted radiation is not plane-pol
arized but elliptically polarized
kd
o
r
l
r
I
I
k
c
k
k






10
from
dichroism
circular
molar l 


19.    
l
A
A R
L
4
303
.
2
cm
rad 1
- 


ellipticity
l path length through the sample
A absorption
Circular Dichroism

20. Circular dichroism
  is therefore the angle between the initial plane of polarization and th
e major axis of the ellipse of the resultant transmitted light
• A quantity  is defined such that
tan  is the ratio of the major and minor axis of the ellipse of the trans
mitted light
 ’ approximates the ellipticity
• When expressed in degrees, ’ can be converted to a specific ellipticit
y [] or a molar ellipticity []
• CD is usually plotted as []
 
   
 
θ
10
0.3032
ε
ε
10
θ
y
ellipticit
molar
d
c'
y
ellipticit
specific
3
r
l
2















M

21. 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 compo
nent leads to ellipticity (CD) an
d optical rotation (OR).

22. The difference between the absorption of left and right
handed circularly-polarised light and is measured as a
function of wavelength. CD is measured as a quantity
called mean residue ellipticity, whose units are degre
es-cm2
/dmol.
Circular Dichroism

23. ORD and CD
• CD plots are Gaussian rather than S-shaped.
• Positive or negative deflections depend on the sign of 
or [] and corresponds to the sign of the Cotton effect
• ORD spectra are dispersive (called a Cotton effect for a si
ngle band) whereas circular dichroism spectra are absorpt
ive. The two phenomena are related by the so-called Köni
g-Kramers transforms.
• Maximum of the CD occurs at the absorption max
• Where more than one overlapping Cotton effect, the CD
may be easier to interpret than the ORD with overlapping
S-shaped bands

24. ORD spectra are dispersive (called a Cotton effect for a singl
e band) whereas circular dichroism spectra are absorptive. T
he two phenomena are related by the so-called König-Kram
ers transforms.

27. Sample Preparation
• Additives, buffers and stabilizing compounds:
Any compound which absorbs in the region of
interest (250 - 190 nm) should be avoided.
• A buffer or detergent or other chemical should
not be used unless it can be shown that the
compound in question will not mask the protein
signal.

28. Sample Preparation
• Protein solution: From the above follows that the
protein solution should contain only those
chemicals necessary to maintain protein stability,
and at the lowest concentrations possible. Avoid
any chemical that is unnecessary for protein
stability/solubility. The protein itself should be as
pure as possible, any additional protein or peptide
will contribute to the CD signal.

29. Sample Preparation
• Contaminants: Unfolded protein, peptides, particul
ate matter (scattering particles), anything that adds
significant noise (or artifical signal contributions)
to the CD spectrum must be avoided. Filtering of t
he solutions (0.02 um syringe filters) may improve
signal to noise ratio.
• Data collection: Initial experiments are useful to e
stablish the best conditions for the "real" experime
nt. Cells of 0.5 mm path length offer a good startin
g point.

30. Typical Initial Concentrations
Protein Concentration: 0.5 mg/ml
Cell Path Length: 0.5 mm
Stabilizers (Metal ions, etc.): minimum
Buffer Concentration : 5 mM or as low as possi
ble while maintaining protein stability