Circular Dichroism

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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 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
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• 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.

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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

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Polarized Light
Linear Polarized Light
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Circular Polarized Light
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 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
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Polarized Light

• Circularly Polarized Light

Left-handed
right-handed
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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 plane-polarized radiation will be rotated
  through an angle ?
• nl, nr are the indices of the refraction for
  left-handed and right-handed polarized light
• ? is in radians per unit length (from ?)

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Optical Rotation
n refractive index l wavelength of light
f 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 ? ??MW?10-2

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Optical rotary dispersion

• 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 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

• 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 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

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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
  region of the appropriate absorption band
• CD is plotted as ?l-?r vs ?
• For CD, the resulting transmitted radiation is
  not plane-polarized but elliptically polarized

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Circular Dichroism
q ellipticity l path length through the
sample A absorption
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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 thattan ? 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 ?

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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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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.
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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
  effect for a single band) whereas circular
  dichroism spectra are absorptive. The two
  phenomena are related by the so-called
  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

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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.
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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 protein
  signal.

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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. 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.

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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.

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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 as possible
  while maintaining protein stability