Transcript Circular Dichroism

Circular Dichroism
Part I. Introduction
Circular Dichroism
Circular dichroism (CD) spectroscopy measures differences
in the absorption of left-handed polarized light versus righthanded polarized light which arise due to structural
asymmetry. The absence of regular structure results in zero
CD intensity, while an ordered structure results in a
spectrum which can contain both positive and negative
signals.
Jasco J-810 Circular Dichroism System
Chiral structure can be distinguished and
characterized by polarized light
Optical rotation: the rotation of linearly polarized light by
the sample
Optical rotary dispersion: the variation
of optical
rotation as a function of wavelength. The spectrum of optical
rotation.
Circular Dichroism: the difference in absorption of left
and right circularly light.
Types of polarized light
• Plane polarized light consists two circularly
polarized components of equal intensity
• Two circularly polarized components are like leftand right-handed springs
• As observed by looking at the source, righthanded 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
Polarized Light
Linear Polarized Light
2
z 
z  ct 
E x ( z , t )  E0 sin 2   t   E0 sin

c 
E y z , t   0
Circular Polarized Light
Passing plane polarized light through a
birefringent plate (in the z-direction)
which splits the light into two planepolarized beams oscillating along different
axes (e.g., fast along x and slow along y).
When one of the beams is retarded by 90º
(using a quarter-wave retarder) then the
two beams which are now 90º out of phase
are added together, the result is circularly
polarized light of one direction. By
inverting the two axes such that the
alternate beam is retarded than circularly
polarized light of the other direction is
generated.
The result of adding the right and left
circularly polarized that passes through the
optically active sample is elliptically
polarized light, thus circular dichroism is
equivalent to ellipticity
Polarized Light
Circularly Polarized Light
Left-handed
 z ct 
E x z, t   E0 sin 2   
  
 z ct 1 
E y z, t   E0 sin 2    
  4
right-handed
 z ct 
E x z, t   E0 sin 2   
  
 z ct 1 
E y z, t    E0 sin 2    
  4
Optical rotary dispersion
• If the refractive indices of the sample for the left
and right handed polarized light are different,
when the components are recombined, the planepolarized radiation will be rotated through an
angle 
• nl, nr are the indices of the refraction for lefthanded and right-handed polarized light
•  is in radians per unit length (from )
nl  nr
α
λ
Optical Rotation

rotation rad cm
-1


   n L  n R 

n
refractive index

wavelength of light

angle of rotation
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
10 2 α
α 
lc
l  pathlength in decimeters
g
c
100 mL
Optical rotary dispersion
α
α 
c' d'
• 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
M   M α10
2
Mα 102

c' d '
• M = molecular weight of the optically active component
• the 10-2 factor is subject to convention and is not always included in [M]
Optical rotary dispersion
M   M α102
Mα 102

c' d '
• 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]
Optical rotary dispersion
• ORD curve is a plot of molar rotation [] or [M] vs 
• Clockwise rotation is plotted positively;
counterclockwise rotation is plotted negatively
• ORD is based solely on the index of refraction
• So-called plain curve is the ORD for a chiral
compound that lacks a chromophore
• Chiral compounds containing a chromophore can give
anomalous, or Cotton effect, curves
Cotton Effect
•Positive Cotton effect is where
the peak is at a higher
wavelength than the trough
•Negative Cotton effect is the
opposite
•Optically pure enantiomers
always display opposite Cotton
effect ORD curves of identical
magnitude
•Zero crossover point between
the peak and the trough closely
corresponds to the normal UV
max
Circular Polarized Light
Circular Polarized Light
Circular dichroism
• Measurement of how an optically active compound
absorbs right- and left-handed circularly polarized light
• All optically active compounds ex-hibit CD in the region
of the appropriate absorption band
• CD is plotted as l-r vs 
• For CD, the resulting transmitted radiation is not planepolarized but elliptically polarized
kl  k r
molar circular dichroism   l   r 
c
 kd
k from I  I o10
Circular Dichroism

 rad cm
-1

2.303  AL  A R 

4l

ellipticity
l
path length through the sample
A
absorption
Circular dichroism
•  is therefore the angle between the initial plane of polarization
and the 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
transmitted light
• ’ approximates the ellipticity
• When expressed in degrees, ’ can be converted to a specific
ellipticity [] or a molar ellipticity []
• CD is usually plotted as []
specific ellipticit y    

c' d
2




molar ellipticit y  θ  M  10
ε l  ε r  0.3032 103 θ
Linear polarized light can be
viewed as a superposition of
opposite circular polarized
light of equal amplitude and
phase
different absorption of the leftand right hand polarized
component leads to ellipticity
(CD) and optical rotation (OR).
Circular Dichroism
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
degrees-cm2/dmol.
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
single band) whereas circular dichroism spectra are
absorptive. The two phenomena are related by the socalled König-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
ORD spectra are dispersive (called a Cotton effect for a
single band) whereas circular dichroism spectra are
absorptive. The two phenomena are related by the so-called
König-Kramers transforms.
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.
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.
Sample Preparation
• Contaminants: Unfolded protein, peptides,
particulate matter (scattering particles), anything
that adds significant noise (or artifical signal
contributions) to the CD spectrum must be
avoided. Filtering of the solutions (0.02 um
syringe filters) may improve signal to noise ratio.
• Data collection: Initial experiments are useful to
establish the best conditions for the "real"
experiment. Cells of 0.5 mm path length offer a
good starting point.
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
possible while maintaining protein stability