Spectroscopy 2003: 5e kwartaal UV Circular Dichroism

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Spectroscopy 2003 5e kwartaal UV Circular
Dichroism

• Dr. Marco Tessari
• Afdeling Fysische Chemie
• This presentation can be found at the website
  http//www.nmr.kun.nl/Education

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Overview
Introduction Optical Activity Polarization of EM
Radiation Experimental Detection of Optical
Activity -Ellipticity -Optical
Rotation -Circular Dichroism (CD) -Circular
Birefringence Application of CD to conformational
studies Material from Cantor and Schimmel
Biophysical Chemistry ch. 8
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IntroductionOptical Activity
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Introduction Optical Activity
So far, our attention was focused on the energy
transfer from EM radiation to matter as a
consequence of such exchange of energy,
molecules are promoted to higher rotational-,
vibrational- and electronic states. We are now
going to examine what effects such EM-matter
interaction can determine on the trasmitted
radiation.
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Introduction Optical Activity
Optical Activity refers to the capacity of some
substances to alter the properties of transmitted
light. In general, for small molecules, optically
activity is a consequence of lack of symmetry.
For macromolecules an important contribution to
their optical activity derives from their
conformation. Therefore, optical activity can
provide important information on their structural
properties in solution.
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Polarization of EM Radiation
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Polarization of EM Radiation
EM radiation is a transverse wave consisting of
oscillating electric- and magnetic fields,
mutually perpendicular. In general, the
oscillation of the electric field does NOT take
place on a fixed (y,x) plane. The radiation is
not polarized.
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Polarization of EM Radiation
When EM radiation is not polarized, all types of
oscillations are equally probable. As a
consequence, the plane of oscillation of the EM
radiation varies randomly in time.
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Polarization of EM Radiation
A polarizing filter can convert unpolarized into
linearly polarized EM radiation. A good example
of this is a Polaroid filter. This kind of filter
is made up of parallel strands of long molecules.
Only the light polarized along a certain
direction passes through the filter, while the
perpendicular component gets completely absorbed.
 
The vertically polarized component of EM
radiation passes through.
Unpolarized EM radiation
The horizontally polarized component of EM
radiation is absorbed
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Polarization of EM Radiation
Linear polarized radiation correspond to the
superposition of left and right circularly
polarized waves of the same intensity.
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Polarization of EM Radiation
Elliptical polarized radiation correspond to the
superposition of left and right circularly
polarized waves of different intensity.
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Experimental Detection of Optical Activity
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Experimental Detection of Optical Activity

• There are four experimentally detectable ways
  that an optically active ABSORBING sample can
  alter the properties of transmitted radiation
• Ellipticity
• Optical Rotation
• Circular Dichroism
• Circular Birifringence

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Ellipticity
In general, an optically active sample will
convert linear polarized into elliptical
polarized radiation. The ellipticity of the light
is one measure of optical activity and it is
defined as
where a and b are the major and the minor axis of
the ellipse, respectively
Linear polarized radiation
Elliptical polarized radiation
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Optical Rotation
The orientation of the ellipse defines the
Optical Rotation (f), a second indicator of
optical activity. The optical rotation as a
function of the wavelength of the radiation is
called Optical Rotatory Dispersion (ORD)
f
Linear polarized radiation
Elliptical polarized radiation
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Circular Dichroism
Linear polarized radiation correspond to the
superposition of left and right circularly
polarized waves. In an optically active medium
these two components are absorbed to a different
extent
This phenomenon is called Circular Dichroism.
Linear polarized radiation
Elliptical polarized radiation
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Circular Birefringence
An optically active sample has two different
refraction index, nL and nR, for the two circular
components of radiation. This means that one of
the two components propagates more rapidly than
the other through the medium. The result is a
phase shift between the two components,
proportional to the refraction index difference
nL nR. This phenomenon is called Circular
Birefringence.
Linear polarized radiation
Elliptical polarized radiation
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Experimental Detection of Optical Activity
Circular Dichroism and Ellipticity refer to the
same physical phenomenon. The same holds for
Optical Rotation and Birefringence. This is
expressed by the followings equations
Usually only Circular Dichroism and Optical
Rotation are measured experimentally for
practical reasons.
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Experimental Detection of Optical Activity
Exercise 1 A sample of 1cm path-length rotates
linear polarized radiation (l300nm) of f 0.01
deg. Calculate its circular birefringence
(defined as nL - nR).
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Experimental Detection of Optical Activity
Exercise 1 Solution A sample of 1cm pathlength
rotates linear polarized radiation (l300nm) of f
0.01 deg. Calculate its circular birefringence
(defined as nL-nR).
Almost undetectable.
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Experimental Detection of Optical Activity
Exercise 2 A sample of 1cm pathlength has an
ellipticity of q 0.01 deg. Calculate its
circular dichroism (defined as AL-AR).
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Experimental Detection of Optical Activity
Exercise 2 Solution A sample of 1cm pathlength
has an ellipticity of q 0.01 deg. Calculate its
circular dichroism (defined as AL-AR).
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Experimental Detection of Optical Activity
The measurement of Circular Dichroism is very
similar to the conventional UV absorption.
Circular Dichroism is measured exposing the
sample alternatively to left-hand and right-hand
circularly polarized light and detecting the
differential absorption. The difference,
typically about 0.03 to 0.3 of the total
absorption, can be accurately determined with
modern instrumentation. CD is usually reported in
ellipticity units, using the expression
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Experimental Detection of Optical Activity
The measurement of Optical Rotation requires a
rather simple instrument called POLARIMETER.
Since most natural compounds are optically
active, Optical Rotation is usually employed to
characterize the purity of products in
pharmaceutical industry, food industry, flavor
industry, etc. A modern polarimeter can detect
optical rotations as little as 0.002 deg.
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Experimental Detection of Optical Activity
To compare results from different samples, it is
necessary to normalize the experimental data with
respect to concentration (c), path length (l)
and, for polymers, number of chromophores (Ncr )
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Experimental Detection of Optical Activity
Exercise 3 A sample of a polypeptide consisting
of Ncr20 amino-acids (optical path l 0.2 cm,
concentration c 10mM) has an ellipticity q
-9.6 mdeg at l 222 nm. The ratio of the molar
extinction coefficients for left and right
circular polarized light eL/ eR 0.964 at l 222
nm. Determine the transmittance of this sample
for linear polarized light at l 222 nm, with an
optical path l 1 cm.
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Experimental Detection of Optical Activity
Exercise 3 Solution
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Experimental Detection of Optical Activity
Exercise 3 Solution
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Application of CD to Conformational Studies
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Application of CD to Conformational Studies
A quantum-mechanical analysis reveals that ORD
and CD spectra of a biopolymer are very sensitive
to its geometry. The ab-initio calculation of CD
spectra of macromolecules is still a task of
formidable complexity. Therefore, a number of
semi-empirical methods have been developed to
extract structural information from CD data. For
proteins and peptides the major objective has
been to deduce the secondary structure from
measured CD spectra. In general, the CD spectrum
of a poly-peptide in the region 190-230 nm is
dominated by the signals of the peptide groups.
The CD spectrum in this region can then be
expressed as the sum of the contribution of
individual secondary structure regions
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Application of CD to Conformational Studies
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Application of CD to Conformational Studies
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Application of CD to Conformational Studies
The decomposition of a CD spectrum in the sum of
the contributions of the three basic
conformations is performed routinely in the
conformational analysis of peptides and proteins.
The accuracy of this method is hardly better than
5-10. A number of factors limit the accuracy of
this approach -the contributions from other
chromophores -the choice of the three basis
spectra -the packing of secondary structure
elements in the tertiary structure. In summary,
this analysis of CD data provides a rapid,
global, but not very accurate picture of the
secondary structure of a polypeptide.
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Application of CD to Conformational Studies
The CD technique is, however, very reliable for
monitoring changes in the conformation of
proteins under different conditions (denaturation
studies, unfolding experiments, binding
experiments, etc). The advantage of CD for
monitoring conformational changes is its
sensitivity. Even if a detailed
structural interpretation is not possible, a
change in structure will almost surely show up as
a change in the CD spectrum.
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Application of CD to Conformational Studies
The CD spectra of the three basic conformations
(alpha-helix, beta-sheet and random coil) are
very different from each other. Therefore, minor
local conformational rearrangements are reflected
in noticeably changes in the CD spectrum.
alpha-helix
alpha-helix
beta-sheet
random coil