A brief presentation about optical rotatory dispersion and circular dichroism

2. • INTRODUCTION
• FUNDAMENTALS IN ORD
• OPTICAL ROTATORY DISPERSION
• COTTON EFFECT
• CIRCULAR DICHROISM
• INSTRUMENTATION OF ORD AND CD
• HYPHENATED TECHNIQUES
• DIFFERENCE BETWEEN ORD AND CD
• APPLICATIONS
• REFERENCES

3. LIGHT
Oscillations of electromagnetic field
Electric field Magnetic field
Study of light is called “OPTICS” 1

4. Study of interaction between matter and
electromagnetic radiations
Interacts with
light
Matter
(solid , liquid
, gas)
2

5. OPTICAL ROTATORY CIRCULAR
DISPERSION DICHROISM
Rate of change in optical rotation with difference in absorption between
respect to wavelength left handed and right handed
circularly polarised light as a
function of wavelength
3

6. Circular birefrigence
Optical activity Specific
rotation
Plane polarised light
FUNDAMENTALS
4

7. NATURAL LIGHT : Natural light is having two
components electric and magnetic component both are
perpendicular to each other
POLARISED LIGHT : polarised light having electric and
magnetic components confirmed to certain particular
directions
5

8. Linear or plane Circular Elliptically
Polarised light Polarised light Polarised light
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9. The compounds which are having ability to rotate plane polarised
light(PPL) are called optically active compounds
This property of compound is called optical activity which is
measured by polarimeter.
Compounds which rotates PPL to right are dextrorotatory (+)
and to left are levorotatory(-)
7

10. Specific rotation, [α], is a fundamental property of chiral
substances that is expressed as the angle to which the
material causes polarized light to rotate at a particular
temperature, wavelength, and concentration.
8

11. BI REFRINGENCE
2 REFRACTION
It is the difference in refraction ( and associated speed of light) of
left and right circularly polarised light
9

12. Linear circular
It is an optical property that It is an optical property that
involves unequal refraction involves unequal refraction of
linear polarised light in 2 LCPL and RCPL caused by
orthogonal planes chiral substance
10

13. • It is defined as dependence of optical rotation on
wavelength.
• Unmatched left and right polarizations (LPL &RPL)
results in ORD
SPEED VL ≠ VR
WAVELENGTH λL≠λR
ABSORBANCE εL≠εR
REFRACTIVE INDEX rL≠rR
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14. • Plot of specific rotation as a function of wavelength
12

15. • ORD curve is a plot of molar rotation [α] or [M] vs λ
• Clockwise rotation is plotted positively;
counterclockwise rotation is plotted negatively
• ORD is based solely on the index of refraction
• A 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
13

16. • 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 λmax
14

17. Linear dichroism circular dichroism
“Dichroism” is used to denote direction-dependent
light absorption.
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18. • Linear dichroism : Linear dichroism refers to the
differential absorption of light polarized parallel or
perpendicular to the some reference axis.
• circular dichroism: only occurs at wavelengths of light
that can be absorbed by a chiral molecule. At these
wavelengths Left‐and right‐circularly polarised light will
be absorbed to different extents. For instance, a chiral
chromophore may absorb 90% of RCPL and 88% of
L‐CPL. This effect is called circular dichroism and is the
difference in absorption of L‐CPL and R‐CPL. Circular
dichroism measured as a function of wavelength is
termed circular dichroism (CD) spectroscopy 16

19. Molar differential extinction coefficient:
Δε = Difference between molar extinction coefficients of
left and right polarised lights
Molar ellipticity:
Δε = εL – εR
ε = 3300 Δε
CD spectrum is a plot of molar ellipticity as a function of
wavelength.
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20. Circular dichroism exhibited because of :
Dextro rotatory components
Levo rotatory components
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21. At present three commercial instruments, the Cary,
the Jasco, and the Jouan, are capable of yielding
high resolution CD and/or ORD spectra with
relatively low noise levels down to about 185 nm,
and are suitable for protein studies.
19

22. • Circular dichroism (CD) spectroscopy measures differences in the absorption
of left-handed polarized light versus right-handed polarized light that 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.
• CD can be used for:
• Determination if a protein is folded
• Characterization of secondary structure (α-helix, β-sheet)
• Studying conformational stability of proteins:
• pH stability
• buffers
• temperature
• addition of stabilizers 20

23. •Characterization of secondary structure is probably
the most used CD spectroscopy application.
Secondary structure can be identified in the "far-UV"
spectral region (190-250 nm). The protein peptide
bond is the chromophore, and it is possible to detect a
signal if the protein is in a specific secondary
structural conformation (α-helix, β-sheet).
•Alpha-helix, beta-sheet, and random coil structures
each give rise to a characteristic shape and magnitude
of CD spectrum
21

24. • JASCO offers a range of measurement modes and
hyphenated techniques
• Circular dichroism (CD) and UV-Vis absorbance (standard)
• Scanning emission fluorescence and Fluorescence detected
CD
• Stopped-flow CD, Stopped-flow absorbance, Stopped-flow
fluorescence
• High throughput CD (HTCD)
• Chiral HPLC detection (LCCD) .
• Near infrared CD (NIRCD)
• Linear dichroism (LD) and Optical rotatory dispersion
(ORD)
22

25. ORD CD
Dispersive Absorptive
Differential speed of LCPL AND RCPL
( CL ≠ CR )
Differential absorption of LCPL AND
RCPL (εL ≠ εR )
Causes rotation of plane polarised
light
Gives elliptically polarised light
Occurs at all wavelenghts Occurs only at characteristic
wavelengths
To determine concentration To determine secondary structure
ORD graphs are plotted between
specific rotation and wavelength
CD graphs are plotted between molar
ellipticity and wavelength
23

26. CD/ORD instruments are now used extensively in a number of application
areas:
• Protein folding studies
• Protein conformational studies
• DNA/RNA interactions
• Enzyme kinetics
• Organic stereochemistry studies
• Purity testing of optically active substances
• Quantitative analysis of pharmaceuticals
• Natural organic chemistry
• Biochemistry and macromolecules
• Metal complex chemistry
• Polymer chemistry
• Medical sciences
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27. • CIRULAR DICHROISM THEORY AND INSTRUMENTATION
A. ABU-SHUMAYS and JACK J. DUFFIELD Applied Physics
Corporation, Monrovia, Calif.
• Circular Dichroism and Optical Rotatory Dispersion of Proteins and
Polypeptides.
By ALICE J. ADLER, :NORMA J. GREENFIELD, and GERALD D.
FASMAN.
• Difference Optical Rotatory Dispersion and Circular Dichroism
By JEN TsI YANG and KvE HVNG CHAr.
• J-815 Circular Dichroism Spectrometer Leading the development
of chiroptical instrumentation
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