optical rotatory disp. cotton effect

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Smt. Kishoritai bhoyar college of pharmacy, new kamptee. : 

Smt. Kishoritai bhoyar college of pharmacy, new kamptee. Optical rotatory dispersion(ORD) and circular dichroism(CD) BY GUIDED BY BHAVIK S.KOTAK DR. K.R.GUPTA

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INTRODUCTION:- NATURAL LIGHT: Natural light is having two components electric and magnetic component, and both are perpendicular to each other in different planes. POLARIZED LIGHT: Polarized light have electric and magnetic components confirmed to certain particular directions UNPOLARISED LIGHT POLARISED LIGHT

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Types of polarized light. Linear or plane polarized light. Circular polarized light. Elliptically polarized light, Linear polarized light:- If a beam of polarized light proceeding in a straight line has all its vibrations in one plane is called Linear polarized light Circular polarized light:- when vibration are along a circle lying in a plane perpendicular to the direction of propagation the light is called circular polarized light Elliptically polarized light:- when vibration are along a ellipse lying in a plane perpendicular to the direction of propagation the light is called elliptically polarized light

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CONVERSION OF LIGHT INTO CIRCULAR POLARIZED LIGHT:- Passing plane polarized light through a birefringent plate (in the z-direction) which splits the light into two plane-polarized beams oscillating along different axis (e.g., fast along x and slow along y). When one of the beams is retarded by 90º (using a quarter-wave retarder) then the two beams which are now 90º out of phase are added together, the result is circularly polarized light of one direction. By inverting the two axis such that the alternate beam is retarded than circularly polarized light of the other direction is generated.

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For the conversion of light in to a circular polarized light two steps must be considered Conversion of natural light to plain polarized light Polarized beam must be passed through some device capable of converting it to circular polarized light Fresnel rhomb :- it is less expensive and retardation is based on the refractive indices. It depends on internal reflections. Pockels type :- it is highly expensive and retardation can be selected by suitable potential. It is electro-optical modulator in which a high potential in kv range is applied across a plane of potassium dihydrogen phosphate or similar piezoelectric crystal cut perpendicular to its optical axis.

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PLANE OF VIBRATION The plane containing the direction of vibration as well as the direction of propagation is known as plane of vibration WHY ROTATION OCCURS IN POLARISED LIGHT ? In case of optically active the components are retarded to different extents because the refractive indices of the medium for left circularly polarized light(nL) and right circularly polarized light (nR)differ. As a result of this circular birefringence (Δn=nL-nR)the beam emerges form the medium is still plane-polarized but with the plane of polarization inclined at an angle α degrees to the plane of polarization of the incident beam. In case of optically inactive components both circularly polarized components are retarded to the same extent and beam emerges form the medium polarized in the same plane as the incident beam

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Magnitude of rotation depend upon the following factors:- Nature of the substance & solvent Length of the liquid column (l)through which light passes. Concentration of the solution. Temperature of the solution (t). Wavelength of the light used(λ). The presence of traces of other optically active substances. $ Nature of solvent can also effect the results Eg:- 20%w/w solution of nicotine in chloroform has [α]20D = +4o while the same concentration in water give [α]20D = +19o . Chloramphenicol gives a change in sign when rotation is detected in ethanol and ethyl acetate. $ The specific rotation refers to a light path of unity (100mm), but the actual path of the measurement is not important because the rotation increses preciesly linearly with the light path, and the data are readily convertable. Eg:- With low values of rotaion, the use of a 200 mm tuve is usual which will then exactly double the measured value.

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$ The conc however, may have a somewhat non linear effect. Therefore, the concentration range is usually indicated to which a given value of [α] refers $ Temperature changes have several effects upon the rotation of a solution or a liquid. [α]t=[α]20+z(t-20) where, Z= temperature coefficient of rotation T=temperature in degree Celsius. $ wavelength is an important factor, as the slope of the ORD, [α] vs λ, which may be quite steep. Earlier meaurements were only taken at 589 nm as the sodium fames and arc lamps were the only convenient sources of monochromatic light. In recent years, the mercury arc has been used widely and the 546nm 436nm has been isolated by filters. These have the great advantage that the values of [α] are usually substantially greater at the shorter wavelength. $ In some compounds, such as sugars, mutarotaion may be present. These compound exist in two optically active forms at equilibrium. When the compound is dissolved, a considerable time may take place in establishment of the equilibrium. In such cases, the initial and final rotation are noted.

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IMPORTANT TERMINOLOGIES:- SPECIFIC ROTATION Specific rotation is defined as number of degrees of rotation of the plane polarized light produced by one decimeter in length filled with a solution having one gram of substance per ml. the measurement is carried out at a particular temperature using sodium light. The specific rotation can be calculated by following relation

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Faraday effect:- Any liquid or solution when placed in a magnetic field, rotates the planes of polarized light because of the effect of the magnetic field upon the motion of the electrons in the molecules. This effect was discovered by faraday so called faraday effect. Ф=vbl v= verdet constant, b= magnetic induction,l = pathlength KERR EFFECT:- If an optically isotropic substance is kept in an electric field E, the substance become doubly refracting, i.e. Is the refractive index parallel to the direction of the field n|| is not equal to that perpendicular to the field n┴. The difference of these tow qunatities is given as follows: n|| - n┴ = KE2 λ ASYMMETRIC MOLECULE The carbon having four different groups are called as asymmetric molecule. Dissymmetric molecule The molecule lacking an improper axis is called dissymmetric molecule SYMMETRIC MOLECULE The molecule having an improper axis is called symmetric molecule ENANTIOMERS Those optical isomers which are mirror images of each other are called enantiomers

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DIASTEROMERS The isomers which are not mirror images of each other and rotate the plane of ploraized light by different amount and have different physical and chemical properties are called diasteromers. RACEMIC MODIFICATION A mixture of optically active isomer giving rise to an inactive solution is called a RACEMIC MODIFICATION. MOLAR ELLIPTISITY The magnitude of circular dichroism is expressed by the ellipticity, Ф, in radians and is defined by the expression Ф = π ( nl - nd ) λ SCIENTIST AND THEIR CONTRIBUTION JEAN BIOT-------- Suggested optical rotation arises due to asymmetric or chiral centre AIME COTTON-- Discovered Circular Dichroism (CD) VANT HOFF AND LE BELL Pointed out connection between optical activity and molecular asymmetry DRUDE--- First attempt was made to explain optical activity. He has shown that the specific rotation may be expressed as a function of wavelength.

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OPTICAL ROTATORY DISPERSION (ORD) The specific rotation [α], changes with wavelength, is called optical rotatory dispersion (ORD) OR The rate of change of specific rotation with wavelength is called Optical rotatory dispersion OR 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 and angle α Drude has shown that the specific rotation may be expressed as a function of wavelength by an equation [α]= k1 + k2 + k3 +…. λ2- λ21 λ2- λ22 λ2- λ23 where, λ=wavelength of measurement k1,k2,k3 are constants and can be identified with wavelength of maximum absorption

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The relationship between rotation and concentration of a solution is not strictly linear, so that the specific rotation. The concentration of the measurement should always be stated when [α] is given. The relation between [α] and concentration be expressed by one of the following equations proposed by Biot. [α]=A+Bq (1) [α]=A+Bq+Cq2 (2) [α]=A + Bq (3) C+q Where, q is the percentage of solvent in solution and A,B,C, are constants. Equation (1) gives straight line, Equation (2) gives parabola, Equation (3) a hyperbola.

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A represents the plain positive ORD curve that is , the specific rotation increases with decreasing wavelength, and B represents the plain negative ORD curve. The word plain implies that there exist no maximum or minima in the curve. ORD CURVE is a plot of specific rotation or molecular rotation vs wavelength

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COTTON EFFECT A.COTTON was investigating the optical activity of potassium chromium tartarate solution, within absorption bands. Using solutions of potassium chromium tartarate, cotton have shown that RCP(right circular polarized light) and LCP(left circular polarized light) are absorbed to a different extent i.e. Єl ≠ Єd Cotton effect have connection with chromophoric absorption in optically active compounds. “AN ACTIVE SUBSTANCE IS CAPABLE OF EXHIBITING COTTON EFFECT ONLY IN ITS OWN ABSORTION BAND AND ALSO IF THE ELECTRONIC SYSTEM RESONSIBLE FOR THE BAND CONTRIBUTES TO THE OPTICAL ACTIVITY.”

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There will be no cotton effect in the absorption band of an added inactive material. The angle of rotation becomes and is reversed in sign in an absorption band only when the electron or electronic system producing that band is sole, or by far the most important contributor to the total optical activity. If not so , the dispersion curves show the same inflection but the rotation does not pass through zero in the middle ORD and CD studies often provide spectral details for optically for optically active compounds that are absent in their UV spectra.

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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 max

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INSTRUMENTATION FOR ORD:- Light source Monochromator Polarizer Sample holder Analyzer Detector There are two types of main methods available for it they are as follows Null point :- Most of the instruments used are on this principle Ratio method

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AUTOMATIC RECORDING SPECTROPOLARIMETERS:- RUDOLPH SPECTROPOLARIMETER It works on the null point principle, the null point being ascertained by an imposed mechanical oscillation of the analyzer, whose mean angular position is orthogonal with respect to the plane of polarization of the entering light beam. At this point the angular changes +Є and – Є produce the same current in phototransducer system. Fig. 21.13, page no 2.477, Instrumental method of chemical analysis, by G.R. CHATWAL & S.K.ANAND

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CARY SPECTROPOLARIMETER It is similar in principle to the Rudolph instrument, except that in the CARY instrument the mechanical oscillation of the analyzer is replaced by an oscillating brought on by a magneto-optical effect. Faraday cell is placed ahead of analyzer for this purpose. Fig 21.14, page no. 2.477, Instrumental method of chemical analysis by G.R.CHATWAL & S.K.ANAND

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Application of optical rotator dispersion (ORD) QUANTITATIVE ANALYSIS:- As Ord curves do not exhibit the kind of detailed structure as do the IR or NMR spectra, they are not routinely used in compound identification. For highest sensitivity, [α] is used at either maxima or minima to compare the unknown with the standard. EQUILIBRIUM STUDIES If an optically active chromophore takes part in a reaction the extent of the reaction can sometimes be followed by observing the reduction of the cotton effect. Eg:- if hydrochloric acid is added to a solution of (+) 3-methylcyclehexanone in methanol, the cotton effect gets reduced by 93% because of dimethy ketal formation. In alcohol only 33% reduction and in isopropyl alcohol there occurs no reduction.

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CONFORMATION STUDIES The chair and the boat conformation of cyclohexane, the two chair forms which are interconvertible in mobile cyclohexanes and the axial and euqatorial conformation of substituents in stereo chemistry have been well studied by making use of ORD curves. When a carbonyl group in cyclohexane gives rise to uv absorption, asymmetry in the molecule will cause a cotton effect ORD curve. The applicatin of ORD curves in conformational analysis is in protein and polypeptide chemistry. It has been found that α-helix and the percentage of α-helical conformation of the long chain molecule can be redtermined from the ORD curves.

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DETERMINATION OF ABSOLUTE AND RELATIVE CONFIGURATION:- The L-α-aminoacid are by no means all laevorotatory at the medium D line which has been used. Consequently unattractive symbol L(+) and L(-)have been used. Using copper complexes, which have sbsoprtion in the visible region, it has been observed that L(+) valine and L(-) phenyl alanine are essentially identical, despite the chance opposite rotation at 589nm of the parent compounds. This proves identical configuration at the α-carbon atom. The cotton effect of the free amino acids around 225nm are now much used with the help of modern instruments. The determination of absolute configuration is of great importance in natural product chemistry. The carbonly group is paritcularly suited for ORD studies. Cafestol found in coffee beans can be degraded to corresponding ketone. The ORD curve of corresponding ketone and 4-ethlycholestan 3- one reveal that these two molecules must have opposite configuration.

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CIRCULAR DICHROISM Circular dichroism is the differential absorption of left and right handed circularly polarized light The electric field of a light beam causes a linear displacement of charge when interacting with a molecule, whereas the magnetic field of it causes a circulation of charge. These two motions combined result in a helical displacement when light impinges on a molecule (both field vectors in the same place are of the same direction but at different moments of time.)

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Since circularly polarized light itself is a chiral it interacts differently with the chiral molecules, that is two types of molecules interacts differently with chiral molecules. In CD experiment equal amounts of left and right circularly polarized light of a selected wavelength are alternately radiated into a chiral sample. The magnitude of circular dichroism is expressed by the ellipticiy, Ф, in radians and is defined by the expression Ф = π ( nl - nd ) λ

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DELTA ABSORBANCE At a given wavelength ΔA=AL-AR Where, ΔA is the difference between absorbance of left circularly polarized(LCP) and right circularly polarized light(RCP) (usually measure) MOLAR CIRCULAR DICHROISM It can also be expressed by applying beer’s law as ΔA=(ЄL- ЄR)Cl Where, ЄL and ЄR are the molar extinction coefficients for LCP and RCP light. C=molar concentration L = length of path in cm Then, ΔЄ=ЄL-ЄR Is the molar circular dichroism.

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INSTRUMENTS USED IN CIRCULAR DICHRISM An ordinary spectrophotometer can be adapted to measure circular dichroism. It is only necessary to provide some means of production d and l circularly polarized radiation. For this purpose a plane polarized beam can be passed through a quartz-wave plate. If the plate is rotated from -45o to +45o , first d and l circularly polarized light is produced

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APPLICATIONS OF CIRCULAR DICHROISM (CD) SPECTROSCOPY PROTEIN CONFORMATION:- The CD spectrum of a protein can provide a information about the relative amounts of the major types of secondary structure within the protein in solution. CD spectra of the α-helix β conformation and the random coil of poly-L-amino acids are distinctly different form each other. NUCLEIC ACID CONFORMATION:- The CD spectrum of a single stranded nucleic acid may be calculated fairly accurately form a knowledge of its nearest neighbor frequency. Thus any differences between the calculated and measured CD spectrum must be due to variation in structure, such as double-strandedness. The CD spectra are frequently used to study changes in the structure of nucleic acids such as: the los of hleicity of single stranded nucleic acids as a function of temperature or pH structural changes on binding cation and proteins.

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DIFFERENECES BETWEEN ORD AND CD

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COMPARISON OF GRAPH OF ORD AND CD ORD CURVE OF CAMPHOR CD CURVE OF CAMPHOR

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REFERENCES:- B.K.SHARMA, Instrumental method of chemical analysis, 26TH edition, page no:-M-286 to M-307. G.R.CHATWAL & S.K.ANAND, Instrumental method of chemical analysis, 5TH edition page no:- 2.468-2.481. E.L.ELIEL , Stereochemsitry of carbon compounds, Mc-Graw Hill page no:- 398-433. www.wikipedia.org.

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THANK YOU