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8/2/2019 ORD and CD-Very Good

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Circular Dichroism

Part I. Introduction


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Circular Dichroism

Circular dichroism (CD) spectroscopy measures differences

in the absorption of left-handed polarized light versus right-

handed polarized light which arise due to structuralasymmetry. 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.

Jasco J-810 Circular Dichroism System


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Chiral structure can be distinguished and

characterized by polarized light

Optical rotation: the rotation of linearly polarized light by

the sampleOptical rotary dispersion: the variation of opticalrotation as a function of wavelength. The spectrum of opticalrotation.

Circular Dichroism: the difference in absorption of leftand right circularly light.


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Types of polarized light

Plane polarized light consists two circularlypolarized components of equal intensity

Two circularly polarized components are like left-and right-handed springs

As observed by looking at the source, right-handed circularly polarized light rotates clockwise

Frequency of rotation is related to the frequency ofthe light

Can be resolved into its two circularly polarizedcomponents

When added together after passing through anoptically isotropic medium, plane polarized light

results


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Polarized Light

Linear Polarized Light

0,

2sin2sin),( 00

tzE

ctzEtczEtzE

y

x


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Passing plane polarized light through a

birefringent plate (in the z-direction)

which splits the light into two plane-polarized beams oscillating along different

axes (e.g., fast along x and slow along y).

When one of the beams is retarded by 90

(using a quarter-wave retarder) then the

two beams which are now 90 out of phase

are added together, the result is circularly

polarized light of one direction. By

inverting the two axes such that the

alternate beam is retarded than circularly

polarized light of the other direction is

generated.

The result of adding the right and left

circularly polarized that passes through the

optically active sample is elliptically

polarized light, thus circular dichroism is

equivalent to ellipticity

Circular Polarized Light


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Polarized Light

Circularly Polarized Light

4

12sin,

2sin,

0

0

ctzEtzE

ctzEtzE

y

x

4

12sin,

2sin,

0

0

ctzEtzE

ctzEtzE

y

x

Left-handed right-handed


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Optical rotary dispersion

If the refractive indices of the sample for the leftand right handed polarized light are different,when the components are recombined, the plane-polarized radiation will be rotated through anangle

nl, nr are the indices of the refraction for left-handed and right-handed polarized light

is in radians per unit length (from )

nn

rl


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Optical Rotation

RL nn

1-cmradrotation

nrefractive index

wavelength of light

angle of rotation


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Optical Rotation

Usually reported as a specific rotation [],

measured at a particular T, concentration and

(normally 589; the Na D line) Molar rotation [] = []MW10-2

mL100

gc

decimetersinpathlengthlc

10

2

l


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d'c'

Concentration of an optically active substance, c, expressed in g cm-1

(as density of a pure substance)

d = thickness of the sample in decimeters

''

1010

22

dc

MMM

M= molecular weight of the optically active component

the 10-2 factor is subject to convention and is not always included in [M]


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''

1010

22

dc

MMM

M= molecular weight of the optically active

component

n. b. the 10-2 factor is subject to convention

and is not always included in [M]


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ORD curve is a plot of molar rotation [] or [M] vs Clockwise rotation is plotted positively;

counterclockwise rotation is plotted negatively ORD is based solely on the index of refraction

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


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Cotton Effect

Positive Cotton effect is wherethe peak is at a higher

wavelength than the trough

Negative Cotton effect is the

oppositeOptically pure enantiomers

always display opposite Cotton

effect ORD curves of identical

magnitudeZero crossover point between

the peak and the trough closely

corresponds to the normal UV

max


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Circular dichroism Measurement of how an optically active compound

absorbs right- and left-handed circularly polarized light

All optically active compounds ex-hibit CD in the regionof the appropriate absorption band

CD is plotted as l-r vs For CD, the resulting transmitted radiation is not plane-

polarized but elliptically polarized

kd

o

rlr

IIk

c

kk

10from

dichroismcircularmolar l


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l

AARL

4

303.2cmrad

1-

ellipticity

l path length through the sample

A absorption

Circular Dichroism


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Circular dichroism is therefore the angle between the initial plane of polarization

and the major axis of the ellipse of the resultant transmitted light A quantity is defined such that

tan is the ratio of the major and minor axis of the ellipse of thetransmitted light

approximates the ellipticity

When expressed in degrees, can be converted to a specificellipticity [] or a molar ellipticity []

CD is usually plotted as []

100.3032

10yellipticitmolar

dc'yellipticitspecific

3

rl

2

M


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Linear polarized light can be

viewed as a superposition of

opposite circular polarized

light of equal amplitude and

phase

different absorption of the left-

and right hand polarized

component leads to ellipticity

(CD) and optical rotation (OR).


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Thedifference between the absorption ofleft and righthanded circularly-polarised light and is measured as a

function of wavelength. CD is measured as a quantity

called mean residue ellipticity, whose units are

degrees-cm2/dmol.

Circular Dichroism


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ORD and CD

CD plots are Gaussian rather than S-shaped.

Positive or negative deflections depend on the sign of or [] and corresponds to the sign of the Cotton

effect

ORD spectra are dispersive (called a Cotton effectfor asingle band) whereas circular dichroism spectra areabsorptive. The two phenomena are related by the so-called Knig-Kramers transforms.

Maximum of the CD occurs at the absorption max Where more than one overlapping Cotton effect, the CD

may be easier to interpret than the ORD withoverlapping S-shaped bands


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ORD spectra are dispersive (called a Cotton effectfor asingle band) whereas circular dichroism spectra areabsorptive. The two phenomena are related by the so-calledKnig-Kramers transforms.


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Sample Preparation

Additives, buffers and stabilizing compounds:

Any compound which absorbs in the region of

interest (250 - 190 nm) should be avoided. A buffer or detergent or other chemical should

not be used unless it can be shown that the

compound in question will not mask the proteinsignal.


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Sample Preparation

Protein solution: From the above follows that the

protein solution should contain only those

chemicals necessary to maintain protein stability,and at the lowest concentrations possible.Avoidany chemical that is unnecessary for protein

stability/solubility. The protein itself should be as

pure as possible, any additional protein or peptidewill contribute to the CD signal.


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Sample Preparation

Contaminants: Unfolded protein, peptides,particulate matter (scattering particles), anythingthat adds significant noise (or artifical signal

contributions) to the CD spectrum must beavoided. Filtering of the solutions (0.02 umsyringe filters) may improve signal to noise ratio.

Data collection: Initial experiments are useful toestablish the best conditions for the "real"experiment. Cells of 0.5 mm path length offer agood starting point.


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Typical Initial Concentrations

Protein Concentration: 0.5 mg/ml

Cell Path Length: 0.5 mm

Stabilizers (Metal ions, etc.): minimum

Buffer Concentration : 5 mM or as low aspossible while maintaining protein stability

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