Review of Circular Dichroism
Absorption of photons by molecules requires that
electrons in the molecule be able to absorb the
energy of the photon and also its momentum (energy
is conserved and momentum is conserved).
Spectroscopic Methods in Biochemistry
4. CD Spectroscopy
Quantum-mechanically,
we talk about the ‘transition
4. Circular Dichroism - Spectroscopy
moment’ of the molecule, which embodies the
The optical rotatory dispersion (ORD) and the circular dichroism (CD) are special variations
displacement
of electrons in conjunction with their
of absorption spectroscopy in the UV and VIS region of the spectrum. The basic principles of
the two methods from
is the interaction
polarized light
optically active substances. If a
excitation
one oforbital
towith
another.
linearly polarized light wave passes through an optically active substance, the direction of the
polarization will change. This change is wavelength dependent. This phenomenon is called
optical rotatory dispersion (ORD). Linearly polarized light waves can be described as a
superposition of two circularly polarized light waves. If a substance absorbs these two circularly
polarized components to a different extent, i.e. if the absorption coefficient for the right
Dichroism is when a molecule is best able to absorb
photons with particular polarity (that matches the
molecule’s
own polarizability).
circularly polarized component of the polarized light is different from the absorption of the
left circularly polarized light, this difference is described as circular dichroism (CD). We will
first discuss the physical properties of light that are relevant for the study of circular dichroism
of the biological substances.
Polymers oriented in this way (such as polaroid) are used to generate
polarized light in which the electrical field vector oscillates in one direction
only.
Figure 4.1. Linear dichroism of oriented molecules.Spectroscopic Methods in Biochemistry, chapter 4
http://www.uni-konstanz.de/FuF/Bio/folding/4-CD
%20Spectroscopy%20r2.pdf
Components of a spectropolarimeter
Eisenberg and Crothers 1979
Plane-polarized light is created by passing the beam through a material
that absorbs all of the light polarized in one direction (here X) and
therefore transmits only the Y-polarized light.
Alternately a Nicol prism can be used to reflect all light polarized in
one direction, transmitting only light polarized perpendicular to that
direction.
NOTE, most reflection is polarized to some extent (hence the utility of
polaroid glasses).
Optical Rotation
100α
[α ] =
lC
λ
temp
angle in degrees
path length (dm),
concentration g/100 ml
20
For α-D-glucose [α]D= +112.2
Eisenberg and Crothers 1979
‘+’ means rotation to the right as look into the beam.
Optical activity is a property of molecules with chiral
centres.
Tetrahydrocannabinol
A sample problem
If a sample of bromobutane measured under standard conditions
has an observed rotation of −9.2°, what is the enantiomeric
excess?
Examples
•
(S)-2-Bromobutane +23.1°
•
(R)-2-Bromobutane −23.1°
•
D-Fructose −92.4°
•
D-Glucose +52.5°
•
D-Sucrose +66.47°
•
D-Lactose +52.3°
•
Camphor +44.1°
•
Cholesterol −31.5°
•
Paclitaxel −49°
•
Penicillin V +223°
•
(+)-Cavicularin +168.2°
•
Hexol bromocamphorsulphonate 2640°[2]
All values are given in units of deg dm−1cm3 g−1.
Optical Rotatory Dispersion
= Optical rotation vs. λ
Due to physics we will not discuss,
the lines are ‘dispersive’ and can be
difficult to deconvolute when they
overlap.
We prefer Circular Dichroism
for structural studies of complicated
macromolecules.
The following exercises allow you to
see for yourself how both Optical
Rotation and Circular Dichroism arise
from differential effects on the two
rotationally polarized components
that make up plane-polarized light. i.e.
one is affected differently than the
other.
λo is the λmax for absorption.
Eisenberg and Crothers 1979
Plane-polarized light can be thought of as the sum of two oppositely
circularly polarized light components. (Sum, left-polarized, right-polarized).
t=b
t=c
t=a
t=0
t=d
As an exercise: complete each time-point
cartoon by drawing in the blue vector and the sum
vector (black) to confirm that the sum of two
oppositely rotating components of the same
amplitude is plane-polarized.
t=g
t=f
t=e
Optical Rotation is the
result of different refractive
indices for the two different
circularly polarized components
of light.
This causes one component to
rotate more slowly than the
other (v=c/n,
n = refractive index).
As the light passes through the
material, one polarization of the
light is delayed relative to the
other. Once they emerge, they
both recover the same speed.
Optical Rotation: After passing through a birefringent material, one
of the two circularly polarized components has been delayed more
than the other. (Sum, left-polarized, right-polarized)
t=b
t=c
t=a
t=0
As an exercise: draw in the red and
blue components after each has
progressed another 45° and then draw in
the sum at each point.
t=d
t=g
t=f
t=e
Circular dichroism
Δε = ε L − ε R
ε is a function of λ therefore so is Δε.
Δε can be either negative or positive (vs. ε which is always positive).
CD only occurs near an absorption band (i.e. ε≠0)
It also only occurs for molecules that are ‘optically active’.
Optical activity: a molecule cannot be superimposed on its mirror
image.
Opposing enantiomers have oppositely signed CD.
11
Circular Dichroism: After passing through a dichroic material, one of two
oppositely circularly polarized light components is weaker than the other
(has been absorbed more due to a higher ε). (Sum, left-pol, right-pol).
t=b
t=c
t=a
t=0
As an exercise: complete each time-point
cartoon by drawing in the blue vector and the sum
(black) vector to confirm that the sum of two
oppositely rotating components of different
amplitudes is elliptically polarized but not rotated.
(Consider that the light has now emerged from
the material and the blue vector does not shrink
further.)
t=d
t=g
t=f
t=e
Solution to sample problem
If a sample of bromobutane measured under standard conditions
has an observed rotation of −9.2°, what is the enantiomeric
excess?
-9.2° is 40% of -23.1° so we have 40%
Examples
excess of R form.
The remainder of the sample is an equal
mix of R- and S- because it contributes
no net optical rotation. Thus we have an
additional 30% R and 30%S. Total R is 70%
and total S is 30 %.
Enantiomeric excess is 40%.
•
(S)-2-Bromobutane +23.1°
•
(R)-2-Bromobutane −23.1°
•
D-Fructose −92.4°
•
D-Glucose +52.5°
•
D-Sucrose +66.47°
•
D-Lactose +52.3°
•
Camphor +44.1°
•
Cholesterol −31.5°
•
Paclitaxel −49°
•
Penicillin V +223°
•
(+)-Cavicularin +168.2°
•
Hexol bromocamphorsulphonate 2640°[2]
All values are given in units of deg dm−1cm3 g−1.
Plane-polarized light can be thought of as the sum of two oppositely
circularly polarized light components. (Sum, left-polarized, right-polarized)
t=b
t=c
t=a
t=0
t=d
Answer
t=g
t=f
t=e
Optical Rotation: After passing through a birefringent material, one
of the two circularly polarized components has been delayed more
than the other. (Sum, left-polarized, right-polarized)
t=b
t=c
t=a
t=0
t=d
Answer (note that the black sum vector
always stays in the same plane although its
magnitude and sign oscillate).
t=g
t=f
t=e
Circular Dichroism: After passing through a dichroic material, one of two
oppositely circularly polarized light components is weaker than the other
(has been absorbed more due to a higher ε). (Sum, left-pol, right-pol).
t=b
t=c
t=a
t=0
t=d
Answer: Copying all the
sum arrows to start at a
single point, we see that
they map out an ellipse.
t=g
t=f
t=e