ORD & CD

Optical Rotatory Dispersion And Circular Dichroism

Introduction:

What is light? What are its properties?

Light is an form of electromagnetic wave

First predicted by james Clark Maxwell(1831-1879)

Experimentally detected by heinrich hertz(1857-1894)

Light wave is transverse (wave propagation is perpendicular to electric & magnetic field oscillations).

Speed of light is maximum in vacuum (=3*108m/s) No information can be sent at a speed more than the speed of light Light can only be studies indirectly, that is, in terms of how it behaves.

Light is just one form of electromagnetic radiation which is having an,

1. Electric component2. Magnetic component

Both components oscillate in space and the planes containing these components are perpendicular to the direction of propagation. According to all available theoretical and experimental evidence, it is the electric vector rather than the magnetic vector of a light wave that is responsible for all the effects of polarization and other observed phenomena associated with light. Therefore, the electric vector of a light wave, for all practical purposes, can be identified as the light vector.

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Plane polarized light:

The waves of normal light vibrate in all directions at right angles to the direction of travel.

When this unpolarized light is allowed to pass through the polarization filter, the light vibrating in a single plane is obtained and is called plane-polarized light.

Optical activity:

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A substance that rotates plane polarized light is said to be “Optically Active”

Specific Rotation:

Observed rotation depends on the length of the cell and concentration, as well as the strength of optical activity, temperature, and wavelength of light.

[α]λT=α (observed)/c*l

Where c; concentration in g/mL

l; length of path in decimeters.

T; temperature.

λ; wavelength of light(sodium lamp).

Eg; α observed rotation at 20oc=1.25o

l the cell length in dm=20cm=2.0dm

c=concentration g/mL=1.00g/mL

[α]=1.25/2.0dm (1g/mL)=12.5.

Molecular rotation:

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Molecular rotation is obtained by multiplying the specific rotation by the molecular weight, M. since large numbers are usually obtained, a common practice is to divide the result by one hundred; thus:

[M] λT =[α]λ

T*M /100 (M; molecular weight of the sample)

Optically Active Molecules:

The substance which possesses the property of optical activity is said to be an optically active compound. Before the rotation of plane-polarized light by optically active molecules, it is useful to first define the criteria for optical activity. To be optical active, a molecule must not possess any one of the following symmetry elements:

Plane of symmetry () Centre of symmetry (i) An improper axis (s)

What Is Plane Of Symmetry?

It divides a molecule in such a way that points (atoms or groups of atoms) on the one side of the plane form mirror images of those on the other side.

What Is Centre Of Symmetry?

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It is the point from which lines, when drawn on one side &produced an equal distance on the other side, will meet identical points in the molecule.

E.g; 2,4-dimethylcyclobutane-1,3-dicarboxylic acid

What Is Improper Axis Of Symmetry?

It is an axis such that a rotation of 3600/n around it followed by reflection in a plane perpendicular to the axis generates a structure indistinguishable from the original molecule.

E.g; α-truxillic acid.

Dextrorotatory – when the plane of polarized light is rotated in a clockwise direction when viewed through a polarimeter.

(+) or (d)

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Levorotatory – when the plane of polarized light is rotated in a counter-clockwise direction when viewed through a polarimeter.

(-) or (l)

Test for optical activity: chiral molecules are optically active

Chiral – not superimposeable on the mirror image (“handedness”)

Achiral – superimposeable on the mirror image; not chiral.

- Compounds with one chiral center will show optical activity.

- Compounds without chiral centers do not normally show optical activity.

- compounds with more than one chiral center may or may not show optical activity depending on whether or not they are non-superimposable on their mirror image (chiral) or superimposable (achiral).

Optical rotatory dispersion:

The electrical field of plane-polarized light (linearly polarized of LP) can be considered to be composed of two components of fixed magnitude rotating on opposite sense to one another

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Right circularly polarized (ER) Left circularly polarized(EL)

In an ordinary medium vectors EL and ER rotate at the same speed and hence E is confined to the x plane. In an optically active medium EL and ER rotate at different speeds and although the light is still plane-polarized, the plane having E now makes an angle α with x-axis.

x

EL E

α ER

y

Rotation of E in an optically active medium

If light enters matter, its properties may change. Namely, its intensity (amplitude), polarization, velocity, wavelength, etc. may alter. The two basic phenomena of the interaction of light and matter are absorption (or extinction) and a decrease in velocity.

Absorption means that the intensity (amplitude) of light decreases in matter because matter absorbs a part of the light.

The decrease in velocity (i.e. the slowdown) of light in matter is caused by the fact that all materials (even materials that do not absorb light at all) have a refraction index, which means that the velocity of light is smaller in them than in vacuum. The refraction index is the ratio of the velocities of light measured in vacuum and in the given material.

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Rotation occurs because in the optically active medium, the refractive index n would be different for the left & right circularly polarized light;

i.e., nL≠nR

And hence the different speed of rotation & the medium is said to be “circularly birefringent”.

In LP the electric field vector changes its magnitude sinusoidlly in a plane along the direction of the propagation.

In circularly polarized light, the magnitude remains the constant but its direction changes continuously in a helical fashion, either in clockwise(RCP) or in an anticlockwise(LCP) direction.

ORD is the specific rotation(α), changes with wave length. The rate of change of specific rotation with wavelength is known as optical rotatory dispersion.

• If the refractive indices of the sample for the left and right handed polarized light are different, when the components are recombined, the plane-polarized radiation will be rotated through an angle a

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

a Is in radians per unit length (from l)

ORD curve is a plot of molar rotation [a] or [M] vs. l

• Clockwise rotation is plotted positively; counterclockwise rotation is plotted negatively

ORD is based solely on the refractive index.

Circular dichroism:

Circular dichroism is a form of spectroscopy based on the differential absorption of left- and right-handed circularly polarized light.

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α=nl−nr

λ


Some materials possess a special property: they absorb left circularly polarized light to a different extent than right circularly polarized light. This phenomenon is called circular dichroism.

Any linearly polarized light can be obtained as the superposition of a left circularly polarized and a right circularly polarized light wave. Therefore, if linearly polarized light traverses a medium that shows circular dichroism, its properties will change because the medium absorbs the two circularly polarized components to a different extent.

circular dichroism makes plane-polarized light elliptically polar.

The E vector of linearly polarized light (also called plane-polarized light has a constant direction and a modulated amplitude. By contrast, the E vector of circularly polarized light has constant amplitude but a modulated direction.

The superposition of the two components is no longer a linearly polarized wave: the resulting field vector does not oscillate along a straight line but it rotates along and ellipsoid path. Such a light wave is called an elliptically polarized light

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CD is plotted as el-er vs. l

For CD, the resulting transmitted radiation is not plane-polarized but elliptically polarized

At a given wavelength,

Where ΔA is the difference between absorbance of left circularly polarized (LCP) and right circularly polarized (RCP) light (this is what is usually measured).

It can also be expressed, by applying Beer's law, as:

where

εL and εR are the molar extinction coefficients for RCP and LCP light, C is the molar concentration l is the path length in centimeters (cm).

Then

is the molar circular dichroism. This is what is usually meant by the circular dichroism of the substance. . Although ΔA is usually measured, for historical reasons most measurements are reported in degrees of ellipticity. Molar circular dichroism and molar ellipticity, [θ], are readily interconverted by the equation,

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Elliptical polarized light is composed of unequal contributions of right and left circular polarized light.

. ORD curves:

Plain curves:These show no maxima and minima, i.e., they are smooth curves, and

May be positive or negative according as the rotation becomes more positive or negative as the wavelength changes from longer to shorter values.

+

+ve

Rotation

-ve _

300nm Wave length 700nm

Cotton effect curves (single):

The Cotton effect is the characteristic change in optical rotatory dispersion and/or circular dichroism in the vicinity of an absorption band of a substance. The rotation rises sharply to a maximum (peak), then quickly drops down to zero near about lmax, goes down to a minimum(trough), and then rises again slowly giving an S-shaped curve. Such an ORD curve is called an anomalous or a cotton effect curve and is said to exhibit a cotton effect (CE). This phenomenon was discovered in 1895 by the French physicist Aimé Cotton (1869-1951s).

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i. Positive cotton effect curve: The peak is at higher wavelength than the trough.

+ Peak

Rotation amplitude

Trough _


ii. Negative cotton effect curve: Here the trough is at higher wavelength than the peak.

+

Peak

Rotation

Trough _


Molecular amplitude (A):

A= [] p-[] t /100 ([] p-[] t; molecular rotation at peak and trough)

iii. Multiple cotton effect curves: This type of cotton effect curve shows two or more peaks and corresponding troughs.

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+

Rotation

_


Applications of ORD and CD:

A more recent method of conformational analysis of steroids makes use of optical rotatory dispersion curves.

ORD curve have been examined mainly for compounds containing a keto group, and the application of this method to the study of keto steroids by Djerassi et al (1957onwards) has proved highly successful in elucidating configurations. The examination of a large number of saturated keto steroids has shown that the sign, wavelengths of the peak and trough, and the amplitude of the ORD curve depend on the position of the keto group in the nucleus. Furthermore, for a given position of the keto group, difference arise in the ORD curve according as the fusion of rings A & B is cis or trans.

ORD data of some ketones

λnm Sign of curve and

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ketone molecular amplitude peak trough

5α-cholestan-3-one5β-cholestan-3-one5α-cholestan-4-one5β-cholestan-4-one5α-cholestan-6-one5β-cholestan-6-one5α-cholestan-7-one

307 267 307 265 307 267 300 278 307 270 308 270 305 275

+65 -27 -94 +3 -78 -77 -26

If the position of the keto group in the steroid is known, the sign and amplitude of the ORD curve will permit a decision to be made about the nature of the configuration of the ring junction.

Eg; 17α-ethinyl-19-nortestosterone (III) on catalytic reduction (ruthenium oxide) followed by oxidation with N-bromoacetamide, gave 17α-ethyl-19-nor-5α-androstan-17β-ol-3one(IV) together with a small amount of 5β-isomer (V). configuration were assigned on the following evidence.

The two possible products will differ only in the nature of the fusion of rings A & B.

Compound (IV) gave an ORD curve that corresponded to the trans A/B 3-keto steroids, whereas the curve of (V) was closely similar to that of 5β-androstan-17β-ol-3-one. Thus A/B is cis in (V)

ORD data can also be used to determine relative and absolute configurations in steroids. One way is to compare the ORD curve of the compound of unknown configuration with curves of analogous compounds whose absolute stereochemistry has been established

An alternative way of using ORD data makes use of the “Octant Rule”

Octant Rule:

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The octant rule is first formulated by moffitt et al (1961) for the correlation of the CE of chiral cyclohexanone derivatives with their absolute configuration. In the simplest form,

• The space around the C=O group is divided into 8 sectors (octants) with the orthogonal planes, A, B and C defined by xy, zy, and xz.

• The vertical plane A bisects the cyclohexanone chair and the horizontal plane B contains C=O moiety and the two attached carbons C-2 and C-6. & plane C is perpendicular to both the planes A & B and intersects the C=O at the midpoint. The midpoint of the C=O is the origin of the co-ordinate system (so we get front four octants & back four octants). Since it is unusual for substituents to lie in front of the oxygen, the front octants are generally unoccupied.

• Thus they divides the space into four quadrants designated as,1. Upper left(UL)2. Upper right(UR)3. Lower left(LL)4. Lower right(LR)

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The sign for each octant is given in fig. These signs are obtained from the sign of the product of the coordinates of any atom.

The Octant Rule states that atoms lying in the, Back upper left & back lower right octants make positive

contribution. Back lower left & back upper right octants make positive

contribution. Atoms lying in any of the three planes make no contribution. The hydrogen atoms are ignored, i.e, their contribution are

insignificant.

“The contribution of an atom to the sign of the ORD curve is the sign of the product of its co-ordinates”

Applications of octant rule:

1. Determination of Conformation:

Since cyclohexanone does not contain a chiral centre, let us now consider 3-methylcyclohexanone, which has a chiral centre at C-3. Two possible orientations are equatorial and axial methyl, and to apply the Octant rule, the two forms are drawn with the carbonyl bond horizontal. If we line these up with the cyclohexanone molecule shown in fig,

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Carbon atoms 1, 2, 4, and 6 lie on axes and so make no contribution, but atoms 3 and 5 make contributions, but since these are equal and opposite in sign, the net contribution is zero.

Thus the only contributor to the sign is 3-(-5) methyl group. The sign of the ORD curve is expected to be positive if the methyl group is

equatorial & negative if the methyl group is in axial. The observed sign is positive and so the orientation is equatorial.

It can be seen from this example that if we know the sign of the ORD curve, we can elucidate the conformation.

Now let us consider 5α-cholestan-6-one. The positions of the atoms in the back octants are as shown in the fig. if we assume that all ring carbon atoms make equal contributions to the sign of the ORD curve, then

Atoms 2, 1, 10 and 19 cancel out atoms 8, 14, 15 and 18, and since atoms 3, 4, 5, 6, 7, 9, and 11 lie on axes, they make zero contributions. Thus, only the remaining, 12, 13, 17, 16 and R make contributions, and since they all lie in the upper right octant, which is negative, the sign of the curve is predicted to be negative. The observed sign is negative, the actual value of a is -78.

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2. Determination of absolute configuration:

Eg; (+)-trans-10-methyldecal-2-one

The diagram for the back octants is shown in the fig. Atoms 1, 2, 3, 5, and 10 and the methyl-carbon atom lie on axes, and so their contribution is zero. Since all the other atom, 6, 7, 8 and 9 lie in the upper left octant, all make a positive contribution to the sign of the curve, which is therefore predicted to be positive. Djerassi et al(1957) observed a positive effect, and so the absolute configuration is the one shown (VI). the mirror image of (VI) is (VIa), and application of the octant rule shows that all the contributing atoms lie in the upper left octant. The sign of the ORD curve for (VIa) is negative (the ORD curves of enantiomers are mirror images of each other.

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CD and ORD with cotton effects:

1. Functional group analysis:

Sometimes the characteristic bands of a functional group in conventional spectroscopy (eg; IR and UV) may be complicated due to overlapping bands. The cotton effect appears approximately at the absorption maximum (λmax) In UV-Visible region and thus provides correct information regarding the functional group. The absorption maxima of a few common chromophores are given in table.

Chromophoric function λmax nm Chromophoric function λmax nm

Ketoneα,β-unsaturated ketonecarboxylic acidα,β-unsaturated acidesternitro compound

280-300330-360215-220250215-220270

Lactoneα,β-unsaturated Lactoneamides and lactumconjugated dienesubstituted phenylsulphoxide

215-235250-260220-235270250-280210

2. Position of a functional group:

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Sometimes the position of the functional group may be ascertained if the skeletal structure of the compound under investigation is more or less common with that

of an appropriate reference molecule. Among the polycyclic compounds steroidal ketones of known absolute configuration have been studied extensively for their chiroptical properties and they often provide important references for poly cyclic compounds with carbonyl chromophores. The shapes of the CD curves of 1-oxo-,3-oxo- and 7-oxo-5α steroids are shown in the fig.

CD curves of 1-oxo-, 3-oxo- and 7-oxosteroids.

The 3-oxo(as well as 2-oxo) derivatives (eg, 2-and 3-oxocholestanone) exhibit a positive cotton effect, while the 1-oxo and 7-oxo derivatives show a negative cotton effect. 11-oxo and 12-oxo steroids both show a positive cotton effect but 12-oxo derivatives much more strongly so.

This fact has been utilized to determine the position of the hydroxyl group in Rubijervine, a steroidal alkaloid (V).

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Rubijervine and rubijervone

It is preferentially oxidized to rubijervone-12(VI), the ORD curve of which resembles that of a 12-oxosteroid rather than that of an 11-oxosteroid.

3. Determination of configuration:

Assignment of configuration by comparison of CD and ORD curves depends on two principles: ORD and CD curves for enatiomeric structures are exact mirror images of each other across the abscissa(wavelength). Secondly, if the three-dimensional structures of two molecules in the immediate vicinity of the chromophore are identical(with respect to configuration) their CD and ORD curves are expected to give cotton effect of the same sign (even if the ORD curves are plain, they would correspond in shape). If the structures are antipodal, the signs of the cotton effects will be opposite. By way of an example, 19-nortestosterone(VII) when reduced with chemical reagents(Li-ammonia) gives an isomer which is diastereomeric with the one octant by catalytic hydrogenation(H2-Ru).

The first isomer has an ORD curve resembling that of a A/B trans-3-oxosteroid , eg, cholestan-3-one(with a positive cotton effect).

The second shows an ORD curve resembling those of 3-oxo-5β-steroids (with a relatively weak negative cotton effect).Thus the isomers have structures (VIII) & (IX) respectively.

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Cotton effects of 5α- and 5β-nordihydrotestosterone4. Study of conformational changes:

If a molecule exists in more than one conformer in a solution, each conformer will have its own ORD or CD curve and the sign and magnitude of the cotton effects will change with the change of conformer population caused either by a change of solvent polarity or by a change of temperature. A typical example is found in (-)-menthone which exists as two conformers (Xa) & (Xb) as shown in fig. in water (a solvent of high polarity), only a positive cotton effect CD is observed; methanol (a solvent of moderate polarity), two cotton effects one positive and the other negative appear; and in isooctane (a non-polar solvent), the negative cotton effect is very much pronounced. In solvents of intermediate polarity, both the CE’s are seen in different proportions. This type of curves having two CD maxima of opposite signs is called “bisignate”. In this particular two CE’s are presumably due to the two conformers in equilibrium, the diequatorial (Xa) which predominates in a polar solvent( probably a solvation effect) and diaxial (Xb) or the twist form (Xc) which predominates in a non-polar solvent. The diaxial conformer in menthone is stabilized by the synergistic operation of a 2-alkyl ketone effect in Xa and 3-alkyl ketone effect in Xb although the function of the polarity of the solvents is not quit clear. The bisignatecurve are commonly observed in halocyclohexanones, e.g;(+) – trans-2-chloro-5- methylcyclohexanone the conformational equilibrium of which is very sensitive to solvent polarity variation of temperature also produces a similar effect. Thus at low temperature, the diequatorial form of menthone predominates but its population decreases as the temperature is raised. The signs of the CE’s may be related to the two conformers by the application of the octant rule.

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CD spectra and conformational changes in ( - ) - menthone with change of solvent polarity

5. Conformation of protein:

CD and ORD spectra provide important information regarding the secondary structures of polypeptides, proteins, and nucleotides. The α-helical and β-sheet forms of proteins show different and fairly large CD extrema around 220nm while the random coil has still different much weaker cotton effect. The formation of α-helix is a cooperative phenomenon in which the large number of weak H-bonds is involved. It does not form if the poly peptide chain is too short and transition of a random coil to a α-helix with increase of the chain length may observed in CD and ORD spectra.

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For quantitative analysis, some reference compounds are necessary, such as the three (α-helix, β-sheet and random coil) forms of poly L-lysine whose ORD and CD curves are known. The α-helix shows strong negative bands (actually two humps near 220nm), the β-sheet (with strong H-bonds) both parallel and antiparallel shows a strong single negative band at 220nm, and the random coil (with out regular H-bonds) shows a weak positive band in this region. The CD of ORD curve of a protein under investigation is compared with these reference curves and calibrated (as regards the percentage of secondary structures) accordingly. Variations in secondary structures in proteins and polypeptides with the change of pH and of solvents of different H-bonds forming ability may also be studied in this way.

One can measure the CD spectrum of a protein and determine how much of each type of secondary structure (-helix, -sheet, random coil) is present.

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CD spectra are frequently used to study changes in the structure of nucleic acids such as: the loss of helicity of single standard nucleic acids as a function of temperature or pH; structural changes on binding cations and proteins; the effect of binding an amino acid to its appropriate tRNA; transitions between single and double standard nucleic acids; structure of rRNA in the ribosome.

Empirical uses of CD (i.e., inferring changes in structure resulting from alterations in solution conditions):

1. Stability:

Melting of the DNA double helix can be monitored at 190 nm, as shown below left (native DNA, solid line; denatured DNA, dashed; constituent nucleotides, dotted).

- Denaturation of a helical protein can be readily monitored at 222 nm (e.g., acid denaturation of myoglobin shown below), or a b-sheet protein at 217 nm.

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2) Kinetics:

The progress of a reaction can be monitored by CD. E.g., the slow conversion of all cis poly-proline to all trans poly-proline is shown at right.

Conclusion:

The rate of change of specific rotation with wavelength is known as optical rotatory dispersion (ORD).

Circular dichroism (CD) is a form of spectroscopy based on the differential absorption of left- and right-handed circularly polarized light.

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ORD and CD applicable to naturally optically active compounds. ORD and CD curves are obtained by plotting specific or molecular rotation

and molecular ellipticity (or differential dichroic absorption) respectively with wavelength.

They show a cotton effect at or near the wavelength of the maximum (UV/Visible).

ORD and CD curves, particularly those showing one of more cotton effects are extremely useful for providing structural and configurational (including conformational) information.

References:

Organic chemistry; volume 2; stereochemistry and the chemistry of natural products. I.L. FINAR

Stereochemistry of organic compounds. D.NASIPURI Instrumental methods of chemical analysis. GURDEEP R.CHATWAL,

SHAM K.ANAND Stereochemistry of carbon compounds. ERNEST L ELIEL www.biology-online.org/dictionary/Plane-polarized_light www.enzim.hu/~szia/cddemo/edemo8.htm en.wikipedia.org/wiki/Optical_rotatory_dispersion en.wikipedia.org/wiki/Circular_dichroism www.chem.ubc.ca/faculty/straus/C305_CD_ORD.pdf www.ap-lab.com/circular_dichroism.htm www.photophysics.com www.biochem.missouri.edu

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http://www.biochem.missouri.edu/

http://www.photophysics.com/

http://www.ap-lab.com/circular_dichroism.htm

http://www.chem.ubc.ca/faculty/straus/C305_CD_ORD.pdf

http://www.enzim.hu/~szia/cddemo/edemo8.htm

http://www.biology-online.org/dictionary/Plane-polarized_light

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ORD & CD