Contents
Polarization of Light
Polarimetry
Principle, Instrumentation and Applications
Optical Rotatory Dispersion
Circular Dichroism (CD) Spectroscopy
Principle, Instrumentation and Applications
Polarization of Light
E (Electric field)
Unpolarized light
(Electric field vector is
randomly oriented)
Plane polarized light
(Electric field vectors are oriented in
just one plane)
How do you generate plane polarized light?
Unpolarized light Nicol Prism Plane polarized light
(Made from Calcite
crystal)
Polarimetry
Polarimetry measures the rotation of polarized light as it passes
through an optically active solution
Polarimeter
Compounds which rotate the plane of polarized light are called optically
active compounds and this property is known as optical activity.
Dextrorotatory (d or +) : If the compound rotates the plane of polarization to
the right(clockwise) it is said to be dextrorotatory
Laevorotatory (l or -) : If the compound rotates the plane of polarization to
the left(anticlockwise) it is said to be laevorotatory
When a plane polarized light passes through a single chiral compound, there will be a rotation of plane
Enantiomers rotate the plane of polarized light by same amount but it opposite direction
Note: Direction of rotation must be
determined experimentally. There is
no correlation with R or S
configurations
Specific Rotation
Allow us to compare samples collected under slightly different
concentrations and path lengths
Usually, the wavelength Sodium D line 589.3 nm
Q: When measured compound A (4.5 mg/100mL) shown an observed
rotation of -1.4o in a polarimeter with tube length of 5 cm. Calculate
the specific rotation of the compound A.
[α] = α/(c x l) = –1.4O / [(4.5/100) x( 0.5 dm)]
= 62.22O
Optical Purity of Compounds
[Link]
22_etext.pdf
Find Out Questions on specific rotation, optical Purity
and enantiomeric excess and learn how to Solve
Polarimetry-Applications
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Polarimetry-Applications
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Optical Rotatory Dispersion
Optical rotatory dispersion (ORD) is the variation in optical rotation of substance with varying
wavelength of light
Some compounds, measuring specific rotation at single wavelength(Sodium D line), may not give
any observable change. So, ORD can be useful
Circularly Polarized Light
Linear/plane polarized light in Linear/plane polarized light in
XZ Plane YZ Plane
A) B)
Combining A and B with equal Combining A and B with equal amplitude and 90o
amplitude and no phase difference
will give linear polarized light in XY
phase difference will give Circular polarized
plane light in XY plane
[Link]
Circularly polarized can be
either left-handed or right-
handed
Circular Dichroism Spectroscopy
Principle
Circular dichroism (CD) spectroscopy is based on
the differential absorption of left and right- handed
circularly polarized components of incident plane
polarized light. This kind of differential absorption
can be observed only if the chromophore is chiral.
A Chromophore can be chiral due to 1) its intrinsic
structure or 2) its attachment to a chiral center in the
molecule or 3) it is placed in an asymmetric
environment.
In CD spectroscopy, one measures either the ellipticity ()
(Figure 1.4) or the differential absorption (A = AL- AR).
Both and A are related by a relation 32 .98 A EL and ER Electric vector of left-handed and right-
handed circularly polarized light, respectively
CD Instrumentation
Photoelastic
modulator Right-handed Circularly Polarized (RCP)
Left-handed Circularly Polarized (LCP)
Pignataro et. al, Molecules 2020, 25, 4854; doi:10.3390/molecules25204854
CD spectroscopy can be used for
studying
Structural characterization of
proteins, DNA and polymers
under different solution
conditions
Protein-protein interactions
Protein stability under different
conditions such in the presence
of denaturants or some other
additives and different pH
conditions
[Link]
Far-UV CD spectra
Secondary structure of proteins can be
estimated from the far-UV CD spectra (180-
250 nm).In this region the chromophore is the
peptide bond. Upon electronic excitation two
types of transitions can occur in peptide bond;
n * (around 210-220 nm) and to *
(around 190 nm). When this chromophore is
arranged in regular ordered structures, it gives
a characteristic spectral signature of those
structures.
Variation in far-UV CD spectra of proteins
can reveal the secondary structural
changes in the proteins under various
conditions. Chen et. al, Scientific Reports | 6:30804 | DOI: 10.1038/srep30804
Near-UV CD spectra
Near-UV CD spectra (250-350 nm) give information about the
tertiary structure of proteins. In this region, the chromophores
are aromatic amino acids (tryptophan, tyrosine and
phenylalanine) and disulfide bonds. Signals in the 255-270 nm,
275-285 nm and 285-305 nm ranges correspond to
phenylalanine, tyrosine and tryptophan, respectively.
Tertiary fold of proteins place these amino acid residues in an
asymmetric environment and thus any disturbance in the
tertiary structure alters the CD signals. Disulphide bonds give Pabbathi et. al,
[Link] 2013, 14,
weak and broad signals in near-UV region. 2441 – 2449