circular dichroism & ord

16
Optical Rotary Dispersion & Circular Dichriosm spectroscopy By-Lovnish Thakur ASU2014010100099 3 rd SEM, Integrated Biotech

Upload: lovnish-thakur

Post on 23-Feb-2017

756 views

Category:

Education


5 download

TRANSCRIPT

Page 1: Circular dichroism & Ord

Optical Rotary Dispersion & Circular Dichriosm spectroscopy

By-Lovnish ThakurASU20140101000993rd SEM, Integrated Biotech

Page 2: Circular dichroism & Ord

Optical rotatory dispersion & Circular Dichroism

Optical rotatory dispersion is the variation in the optical rotation of a substance with a change in the wavelength of light.

For wavelengths that are absorbed by the optically active sample, the two circularly polarized components will be absorbed to differing extents. This unequal absorption is known as circular dichroism.

Page 3: Circular dichroism & Ord

Polarized Light

• Light with oscillations confined to a single plane.

• Top: vertically polarized light

• Bottom: horizontally polarized light

http://www.photophysics.com/polarization.php

Page 4: Circular dichroism & Ord

Circularly Polarized Light

Sum of vertically and horizontally plane polarized light in which the phases differ by a quarter wave

http://www.photophysics.com/polarization.php

Page 5: Circular dichroism & Ord

Circular Dichroism (CD) Spectroscopy

• Difference in absorption between left-handed and right-handed circularly polarized light as a function of wavelength

• Unlike optical rotatory dispersion (ORD) spectroscopy, which only requires a chiral molecule, CD requires a chromophore in a chiral environment.

Page 6: Circular dichroism & Ord

ORD – why isn’t a chromophore required?

All chiral molecules exhibit circular birefringence, basically the rates of propagation of left and right-handed polarized light (which can be added to form plane-polarized light) are different. This changes their relative phase, and therefore the observed angle at the detector.

http://www.photophysics.com/polarization.php

Page 7: Circular dichroism & Ord

Origin of Ellipticity Unit

http://www.photophysics.com/polarization.php

Page 8: Circular dichroism & Ord

Chromophores in Biomolecules

• Proteins– Aromatic sidechains (π-π* ~280 nm, near UV)• Phenylalanine (ε ~ 250 M-1cm-1)• Tyrosine (ε ~ 1000 M-1cm-1)• Tryptophan (ε ~ 5000 M-1cm-1)

– Backbone amide bond (far UV)• n-π* ~210-220 nm (major peak in UV spectrum, (ε ~

100 M-1cm-1)• π-π* ~190 nm

Sensitive to tertiary structure

Sensitive to secondary structure

Page 9: Circular dichroism & Ord

Working

Measurements carried out in the visible and ultra violet region of the electro magnetic ‐ ‐spectrum monitor electronic transitions, and, if the molecule under study contains chiral chromophores then one CPL state will be absorbed to a greater extent than the other and the CD signal over the corresponding wavelengths will be non zero. ‐

Page 10: Circular dichroism & Ord

• There is a source of monochromatic linearly polarised light which can be turned into either left or right circularly polarised light‐ ‐

• A circular dichroism spectrophotometer has a specialised optical element called a photo elastic modulator (PEM), which produce ‐left and then right circularly polarised light at the drive ‐ ‐frequency.

• On the other side of the sample position there is a light detector.

• When there is no circularly dichroic sample in the light path, the light hitting the detector is constant.

• If there is a circularly dichroic sample in the light path, the recorded light intensity will be different for right and left CPL.‐ ‐

Page 11: Circular dichroism & Ord

thz.yale.edu

Page 12: Circular dichroism & Ord

Applications

• A large subset of the use of circular dichroism in biochemistry is in the understanding of the higher order structures of chiral macromolecules such as proteins and DNA

• Mostly it is widely used to study various secondary structural elements of proteins such as the α helix and the β sheet‐ because CD spectra in the far‐UV (below 260 nm) can be used to predict the percentages of each secondary structural element in the structure of a protein.

• Circular dichroism is to compare two macromolecules, or the same molecule under different conditions, and determine if they have a similar structure.

• For the analysis of biopharmaceutical products to confirm that they are still in a correctly folded active conformation.

Page 13: Circular dichroism & Ord

• In this they examined the thermal denaturation of ribonuclease T1 by CD using both the original and expanded sets of reference proteins and obtained more consistent results with the expanded set. The expanded set of reference proteins will be helpful for the determination of protein secondary structure from protein CD spectra with higher reliability, especially of proteins with significant unordered structure content and/or in the course of denaturation.

• The basic principle involved in the analysis of protein CD spectra, and used in the

estimation of secondary structure fractions, is that the protein CD spectrum can be expressed as a linear combination of CD spectra of individual secondary structure components. Spectra of model polypeptides or of a set of reference proteins with known structures are used to determine the component secondary structure spectra.

Page 14: Circular dichroism & Ord

Conclusion

Circular Dichriosm spectroscopy is a very useful technique to study the structure of biomolecule(chiral molecule). And it is most widely used to study secondary structure of proteins.

Page 15: Circular dichroism & Ord

References

• http://naldc.nal.usda.gov/download/31237/PDF

• http://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/CD%20spectroscopy.pdf

• http://www.uic.edu/orgs/ctrstbio/manuals/adler

Page 16: Circular dichroism & Ord