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Circular Dichroism
Part I. Introduction
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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 structuralasymmetry. 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
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Chiral structure can be distinguished and
characterized by polarized light
Optical rotation: the rotation of linearly polarized light by
the sampleOptical rotary dispersion: the variation of opticalrotation as a function of wavelength. The spectrum of opticalrotation.
Circular Dichroism: the difference in absorption of leftand right circularly light.
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Types of polarized light
Plane polarized light consists two circularlypolarized 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 ofthe light
Can be resolved into its two circularly polarizedcomponents
When added together after passing through anoptically isotropic medium, plane polarized light
results
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Polarized Light
Linear Polarized Light
0,
2sin2sin),( 00
tzE
ctzEtczEtzE
y
x
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Passing plane polarized light through a
birefringent plate (in the z-direction)
which splits the light into two plane-polarized beams oscillating along different
axes (e.g., fast along x and slow along y).
When one of the beams is retarded by 90
(using a quarter-wave retarder) then the
two beams which are now 90 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
Circular Polarized Light
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Polarized Light
Circularly Polarized Light
4
12sin,
2sin,
0
0
ctzEtzE
ctzEtzE
y
x
4
12sin,
2sin,
0
0
ctzEtzE
ctzEtzE
y
x
Left-handed right-handed
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Optical rotary dispersion
If the refractive indices of the sample for the leftand right handed polarized light are different,when the components are recombined, the plane-polarized radiation will be rotated through anangle
nl, nr are the indices of the refraction for left-handed and right-handed polarized light
is in radians per unit length (from )
nn
rl
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Optical Rotation
RL nn
1-cmradrotation
nrefractive index
wavelength of light
angle of rotation
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Optical Rotation
Usually reported as a specific rotation [],
measured at a particular T, concentration and
(normally 589; the Na D line) Molar rotation [] = []MW10-2
mL100
gc
decimetersinpathlengthlc
10
2
l
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Optical rotary dispersion
d'c'
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
''
1010
22
dc
MMM
M= molecular weight of the optically active component
the 10-2 factor is subject to convention and is not always included in [M]
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Optical rotary dispersion
''
1010
22
dc
MMM
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]
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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
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Cotton Effect
Positive Cotton effect is wherethe peak is at a higher
wavelength than the trough
Negative Cotton effect is the
oppositeOptically pure enantiomers
always display opposite Cotton
effect ORD curves of identical
magnitudeZero crossover point between
the peak and the trough closely
corresponds to the normal UV
max
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Circular Polarized Light
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Circular Polarized Light
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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 regionof the appropriate absorption band
CD is plotted as l-r vs For CD, the resulting transmitted radiation is not plane-
polarized but elliptically polarized
kd
o
rlr
IIk
c
kk
10from
dichroismcircularmolar l
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l
AARL
4
303.2cmrad
1-
ellipticity
l path length through the sample
A absorption
Circular Dichroism
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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 thetransmitted light
approximates the ellipticity
When expressed in degrees, can be converted to a specificellipticity [] or a molar ellipticity []
CD is usually plotted as []
100.3032
10yellipticitmolar
dc'yellipticitspecific
3
rl
2
M
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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).
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Thedifference between the absorption ofleft and righthanded 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.
Circular Dichroism
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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 effectfor asingle band) whereas circular dichroism spectra areabsorptive. The two phenomena are related by the so-called Knig-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 withoverlapping S-shaped bands
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ORD spectra are dispersive (called a Cotton effectfor asingle band) whereas circular dichroism spectra areabsorptive. The two phenomena are related by the so-calledKnig-Kramers transforms.
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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 proteinsignal.
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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.Avoidany chemical that is unnecessary for protein
stability/solubility. The protein itself should be as
pure as possible, any additional protein or peptidewill contribute to the CD signal.
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Sample Preparation
Contaminants: Unfolded protein, peptides,particulate matter (scattering particles), anythingthat adds significant noise (or artifical signal
contributions) to the CD spectrum must beavoided. Filtering of the solutions (0.02 umsyringe filters) may improve signal to noise ratio.
Data collection: Initial experiments are useful toestablish the best conditions for the "real"experiment. Cells of 0.5 mm path length offer agood starting point.
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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 aspossible while maintaining protein stability