Circular dichroism

1. CIRCULAR DICHROISM
PRESENTED BY- MAHESH KUMAR
ROLL NO. -191506
DEPARTMENT OF
BIOTECHNOLOGY, CUH

2. INTRODUCTION
• This phenomenon was discovered by Jeam-Baptiste Biat,
Augustin Fresnel and Aime‘s cotton in the first of the 19th
century.
• Dichroism- A property peculiar to certain crystals of reflecting
light in two different colours when viewed from two different
directions .
• Definition –
• This is a form of light absorption spectroscopy that
measures the difference in absorbance of a right and left
handed circularly polarized light by a substance rather than the
commonly used absorbance of isotropic light.

3. PRINCIPLE
• This is a type of absorption spectroscopy method based on the
differential absorption of left and right circularly polarized
light.
• It means that this is relies on the principle of differential light
absorption by the sample the way ,the sample absorbs the left
and right circularly polarized light is actually providing us many
structural information about that particular protein and
sample.

4. CIRCULARLY POLARIZED LIGHT
• Reference-http://youtu.be/F8VGbgi1LwQ

5. CONTINUOUS.....
• Unpolarized light- where the waves of light are having random vibration in all
directions.
• Plane Polarized light- When the light vibrations in only one direction or one
plane.
• Circular plane polarized light-when the light vibrations in only making circularly
vibrations or circular path movements.or
• It occurs when the direction of electric field vector rotates about its
propagation direction while the magnitude of vector is remain constant.

6. CONTINUOUS.....
• REFERENCE-http://you.be/F8VGbi1LwQ

7. ELECTROMAGNETIC RADIATION
• Electromagnetic radiation have two parts-
• 1.Electric field vector.
• 2.Magnetic field vector.
• These field vector occillates perpendicular to each
other,also they are perpendicular to the direction of
propagation.Right,also we know this.
• As given picture-
• Reference-https://youtu.be/FHvvEtMRnZQ

8. INSTRUMENTS
• Light source.
• Filter wheel.
• Polarizer.
• Quarter phase plate.
• Sample cuvette.
• Detector.

9. EXPERIMENTAL DIAGRAM

10. LEFT HANDED CIRCULARLY POLARIZED AND RIGHT
HANDED CIRCULARLY POLARIZED LIGHT
• Reference-http://youtu.be/F8VGbi1LWQ

11. EXPERIMENTAL PROCEDURE
• In this process-
• Light comes through the light source.
• This passes through the filter wheel.
• Here this is light which have specific wavelength.
• And then when this light passes through the polarizer ,now this light is unpolarized.
• This will become polarized due to this polarizer.
• When this polarized light will pass through the quarter phase plate,then that time this
will be circularly polarized.
• After all , circularly polarized light will go through the sample cavette.This adsorbs the
light (CPL) differentially.
• Ultimately which light is transmitted then, this is detected by detector.

12. HOW CAN WE MEASURE THE CD?
• Reference-http://youtu.be/F8VGbgi1LwQ

13. ADVANTAGES AND DISADVANTAGE
Advantages
1. Less extensive preparation.
2. Low concentration of sample
required.
3. Microsecond time resolution.
• Disadvantage
• 1.machines are more expensive
than UV.
• 2.certain buffer components
can absorbs strongly in UV far
region and can cause
interference.

14. APPLICATION
• Determination of secondary structure of protein.
• Ph, Solvent and temperature induced structural
changes can be determined.
• Protein folding/ unfolding responses can be
determined.
• Ligand or ion induced structural damages can be
determined.
• Structural features of chiral compounds can also be
determined.

15. SOFTWARE USED IN C.D.
• 1. CDTOOlX –downloadable software packages for processing and analysis of circular dichroism
spectroscopic data.
• It also runs on moc (osx) and Linux platform.
• Used in the study of diverse samples, including protein, nucleic acid and even chiral small molecules.
• 2.CDSSTR program of dichroweb online series-This program gives us percentage of distorted helix ,
regular sheets.
• Algorithm-CONTIN,SELCON3.
• Website-http://dichroweb.cryst.bbk.ac.uk-tThis is used for cd analysis.
• Bestel.elte.hu-used for beta structure analysis.

16. EXAMPLE: PROTEIN SECONDARY STRUCTURE
DETERMINATION
•
• Reference of image-http://youtu.be/FHvvEtMRnZQ

17. CONTINUOUS...
• As the given in first figure- If we see the spectra of circular dichroism,we get a special type of
information.here,the spectra will be different for alpha helix.and also will be different for beta
sheet and random coil.These are structural motiff in secondary structure of protein.these are
signature of those.alpha helix signature will be red.
• Beta structure signature will be green.if we assume that this is structural dichroism spectra.this is
like a standard curve spectra.now ,here ,we take a protein and find out its c.d.spectra.after
getting spectra, yellow dotted line,we see this type of spectra as given in first ,figure.From this
particular spectra, we Gets the information that in this figure , alpha helix is more.

18. CONTINUOUS....
• Explanation of second figure-assume that we have a protein.we first find out the spectra of this
particular protein.we get the spectra of this protein as in the figure given.from this spectra,we find out
that we have more beta sheets.by this c.d.spectra,we can get more information about secondary
structure of protein.

19. EXAMPLE:HOW CAN WE MEASURE THE CD OF DNA?
• Reference- Https://youtu.be/F8VGbgi1LwQ

20. CONTINUOUS..
• Explanation of first figure-This is the example of CD.We can measure the CD for DNA molecule.
• So,on the x-axis,there is wavelength.
• On the y-axis-we are have circular dichroism.
• So, this is for double helical DNA and when you heated up,it denatures,so when you heating up, then
helix nature is lost and since the helix nature is lost,then circular dichroism is decreasing.so,this is the
direct line at normal temperature,the dotted lines are on different high temperature,the dotted line are
on different high temperature where the helical DNA is opening or DNA is losing it’s property,so I said
the circular dichroism is dependent upon the helical nature of the molecule.Helical nature’s means
more value of circular dichroism.So it is self evidence that at the normal temperature,DNA is helix ,so
we are getting good CD.and you can say,the helix of DNA is opening,so,we are having less value of
circular dichroism.

21. CONTINUOUS..
• Explanation of second figure-one more interesting example like on there is a dye which is acrilidine
orange and it can bind to DNA .so we have measured or people have measured the C.D.of this
dye(acrilidine orange).So ,once it is bound to DNA, then,it is showing helix nature and this showing C.D.
• At 0.0 means this is not bound with DNA.So,there is not a good value of ellipticity.at 0.03 means binding
of DNA with acrilidine orange dye, increasing CD,we can say the ellipticity is increasing or the
absorbance is increasing or you can say the C.D. Is increasing.more concentration of acrilidine
orange,bind with DNA means more CD.

22. REFERENCE
• www.nature.com
• www.photophysics.com
• www.sciencedirect.com
• Let’s crack bio exam(You tube).
• Animated biology with arpan(You
tube)
• http://youtu.be/FHVVEtmrnzq