logging in or signing up u.v.- visible spectroscopy bhavesh.gv89 Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 2634 Category: Education License: All Rights Reserved Like it (4) Dislike it (0) Added: January 21, 2011 This Presentation is Public Favorites: 3 Presentation Description Solvent effects Woodward-Feiser Rules for Calculating Absorption Maxima Application of U.V.-Visual spectroscopy Comments Posting comment... By: vannathankandi (1 month(s) ago) Sir. this presentation is very helpful to me. Kindly send this at saheedvk@gmail.com Thank you Saving..... Post Reply Close Saving..... Edit Comment Close By: sudheer28 (2 month(s) ago) nice presentation.............. pl. send this presentation at k_sudheer28@yahoo.co.in address Saving..... Post Reply Close Saving..... Edit Comment Close By: navinmodi (8 month(s) ago) sir i want u r presentation for my seminar purpose. i liked u r presentation. Saving..... Post Reply Close Saving..... Edit Comment Close By: gopanna (10 month(s) ago) hello mr. bhavesh i am interested to download your uv ppt , may i download it. Saving..... Post Reply Close Saving..... Edit Comment Close By: hetalkagarana (11 month(s) ago) Hello,this presentation can be very helpful to me,can u provide me copy of this on chhayakagarana@gmail.com?thank you. Saving..... Post Reply Close Saving..... Edit Comment Close loading.... See all Premium member Presentation Transcript Slide 1: PRESENTED BY Bhavesh G. Vaghasiya M.Pharm 1st year Maratha Mandal’s college of pharmacy, Belgaum. UV- VISUAL SPECTROSCOPY Department of Pharmaceutics Bhavesh.gv89@gmail.com Slide 2: CONTENTS Solvent effects Woodward-Feiser Rules for Calculating Absorption Maxima Application of U.V.-Visual spectroscopy Slide 3: SOLVENT EFFECTS A most suitable solvent is one that dose not it self absorb in the region under investigation. A dilute solution of the sample is always prepared for the spectral analysis. Most commonly used solvent is 95% ethanol as it is cheap and is transparent down to 210 nm. Commercial ethanol should not be used because it is having benzene which absorbs strongly in the UV region. Some other solvents which are commonly used are water, methyl alcohol, cyclohexane, acetonitrile, diethyl ether etc. Slide 4: SOLVENTS USED IN UV SPECTROSCOPY Slide 5: Hexane and other hydrocarbons can be used because these are less polar and have least interactions with the molecule. For ultra-violet spectroscopy, ethanol, water and cyclohexane are generally used. The position and the intensity of absorption maximum is shifted for a particular chromophore by changing the polarity of the solvent. The absorption maximum for the non-polar compounds is the same in alcohol(polar) as well as in hexane(non-polar). The absorption maximum for the polar compounds is usually shifted with the change in polarity of the solvent. Slide 6: α,β- unsaturated carbonyl compounds shows two different shifts. n π* transition (less intense) In such case, the absorption band moves to shorter wavelength by increasing the polarity of solvent. Eg: absorption maximum of acetone is said to 279nm in hexane as compared to 264 nm in water. A B C D Non polar Polar solvent CD>AB n π* Slide 7: π π* transition (intense) In such case, the absorption band moves to longer wavelength by increasing the polarity of the solvent. The dipole interaction with the solvent molecules lower the energy of the excited state more than that of the ground state. Thus, the value of absorption maximum in ethanol will be greater than that observed in hexane. A B C D Polar solvent Non polar AB>CD π π* Slide 8: Choice of solvent It should not itself absorb radiation in region under investigation. It should be less polar so that it has minimum interaction with the solute molecules. The most commonly employed solvent is 95% ethanol. it is cheap, has good dissolving power and dose not absorb radiation above 210 nm. In other word it is transparent above 210 nm. Some other solvents which are transparent above 210 nm are n-hexane, cyclohexane, methanol, water and ether. Slide 9: Benzene, chloroform and carbon tetrachloride cannot be used because they absorb in the range of 240-280 nm. Hexane and other hydarocarbon are sometimes preferred to polar solvents because they have minimum interaction with the solute molecules. The absorbance of solvent should be checked before use because the presence of small amounts of impurities may give rise to appreciable absorption in the range of the sample. Slide 10: UV spectra of phenol in ethanol and in isooctane Slide 11: WOODWARD-FEISER RULES FOR CALCULATING ABSORPTION MAXIMA Introduction Different terms Rules for conjugated dienes, trienes and polyenes Rules for α,β unsaturated carbonyl compound Rule for Aromatic compound Contents :- Slide 12: INTRODUCTION From the study of the ultraviolet absorption spectra of a large number of compounds, Woodward (1941) gave certain rules for correlating λ max with molecular structure. Scott-Feiser (1959): modified rule with more experimental data. The modified rules known as Woodward-fieser rules, can be used to calculate the position of λ max for a given structure by relating the position of λ max with the position and degree of substitution of chromophore. longer the conjugated system, greater is the wavelength of absorption a maximum. The intensity of the absorption also increases with the increase in length of the chromophore. Slide 13: Different Terms Alicylic dienes or dienes contained in an open chain system i.e, where basic unit is butadiene system. 2. Homo-annular conjugated double bonds Are the conjugated double double bond present in the same ring. it is also called as Homodiene. some examples of this type are: Slide 14: 3. Hetero-annular conjugated double bonds Are the double bonds which are present in different ring. for example. 4. Endocyclic double bond The double bond present in a ring only (inside). 5. Exocyclic double bond The double bond is a part of the ring (outside). Slide 15: Woodward – Fieser’s rules for conjugated dienes,trienes,polyenes etc. According to this rules each type of dienes or triens system is having a certain fixed value at which absorption takes place. This constitutes the basic value or parent value. This value of absorption maximum depends upon …… (I) The number of alkyl substitution or ring residues (II) The number of double bonds which extending conjugation (III) The presence s of polar groups such as Cl,Br,OR, are added to the basic value to obtain λ max for a particular compound. Slide 16: The parent values and increment for different substituents / groups (A) Parent value :- 1) Acyclic conjugated diens and Hetero annular conjugated diens 215 nm 2) Homo annular conjugated diens 253 nm 3) Acyclic triens 245 nm 4) Butadiene system 217 nm (B) Increment :- 1) Each alkyl substitution or ring residue 5 nm 2) Exocyclic double bond 5 nm 3) Double bond extending conjugation 30 nm (C) Auxochromes :- -OR 6 nm -SR 30 nm -Cl, -Br 5 nm -NR2 60 nm -OCOCH3 6 nm Slide 17: EXAMPLES 1. Calculate absorption maximum in UV Spectrum of 2,4 Hexadiene. Butadiene system; basic value = 217nm 2 Alkyl substituent(2X5nm) = 10nm Calculated value = 227 nm Observed value = 227 nm Slide 18: 2. Calculate max for 1,4- dimethylcyclohex-1,3-diene Parent value for homoannular ring : = 253 nm Two alkyl substituents : 2 x 5 = 10 nm Two ring residue : 2 x 5 = 10 nm calculated value : = 273 nm observed value : = 263 nm H3C H3C CH3 CH3 Slide 19: 3. Calculate max of following compound Parent value for heteroannular diene : = 215 nm Four ring residue : 4 X5 = 20 nm Calculated value : = 235 nm Observed value : = 236 nm Slide 20: POLY-enes AND POLY-ynes With increase in the number of double bonds in conjugation, the values of absorption maximum as well as intensity increase. The values of absorption maximum as well extinction coefficient in case –(CH=CH)n-where n>3 are given below. max ε max i. n = 3 275 nm 30,000 ii. n = 4 310 nm 76,000 iii. n = 5 342 nm 122,000 etc. Slide 21: If the Polynes contain more than four double bonds, then Fieser-kuhn rules are applied. According to this approach, both λ max and Emax are related to number of conjugated double bonds as well as other structural units by following equations. λ max =114+5M+n(48.0-1.7n)-16.5 Rendo – 10 Rexo Emax = (1.74 x 104)n Where n = No. of conjugated double bonds. M = No of alkyl or alkyl like substituents on conjugated system. Rendo = No of rings with endocylic double bonds in conjugated system. Rexo = No of rings with exocylic double bonds. Slide 22: EXAMPLE In case of Lycopene In this compound only eleven bonds are in conjugation. Thus, n =11. In addition to this eight substituents.[methyl groups & chain residues]. Thus M =8. As there is no ring system there are neither exo nor Endocylic double bonds in this conjugated system. λ max is calculated as follow. λ max =114+5(8)+11[48.0-1.7(11)]-0-0 =476nm the observed value is found to be 476nm(hexanes Emax =19.1*104 Slide 23: Woodward – Fieser’s rules for calculating absorption maximum in α,β-unsaturated corbonyl compounds Woodward and fieser framed certain rules for estimating the absorption maximum for α-β unsaturated carbonyl compounds. The rules were later modified by Scott in 1964. α-β unsaturated ketones are characterized by a strong max in the region 230-260 nm in alcohol and a weak max in the region 315-320 nm. The basic value for α-β unsaturated Acyclic or six membered ketone is 215 nm. Slide 24: Parent value :- 1) α , β , unsaturated acyclic or 215 nm six membred ring Kitone 2) α , β , unsaturated 202nm five membred ring Kitone 3) α , β , unsaturated aldehyde 207 nm (B) Increment :- 1) Each alkyl substitution or ring residue 10 nm at α position 12 nm at β position 18 nm at γ position 5 nm 2) Exocyclic double bond 5 nm 3) Double bond extending conjugation 30 nm 4)Homo anuular conjugated dienes 39 nm The parent values and increment for different substituents / groups Slide 25: (C) Auxochromes :- α – position β – position γ – position -OH 35 nm 30 nm 50 nm -OR 35 nm 30 nm 17 nm -SR - 85 nm - -Cl 15 nm 12 nm - -Br 25 nm 30 nm - -NR2 - 95 nm - -O-COCH3 6 nm 6 nm 6 nm Slide 26: In these compounds, the actual spectra obtained are affected considerably by the nature of the solvent employed. Hence a solvent correction is applied to the calculated value to get the value for that particular solvent. For common solvent the following correction should be applied in computing absorption maxima. water 8 nm methanol 0 nm chloroform -1 nm hexane -11 nm cyclohexen -11nm Slide 27: 1.Calculate λ max for given compound 1. α -β unsaturated ketone 215nm 2. 2 β alkyl substituents(2X12nm) 24nm Calculated value 239nm Observed value 237nm c EXAMPLES Slide 28: Parent value for ,-unsaturated 6 = 215 nm membered cyclic ketone One ring residue at position = 10nm Two ring residue at position 2 x 12 = 24 nm Double bond exocyclic to two ring 2 x 5 = 10nm Calculated value = 259nm Observed value = 256nm 2.Calculate λ max for given compound Slide 29: Aromatic Compounds The formulation of empirical rules is for the most part is not efficient (there are more exceptions than rules) If the group attached to the ring bears n electrons, they can induce a shift in the primary and secondary absorption bands Slide 30: Electron-donating and electron-withdrawing effects Electron donating Electron withdrawing Slide 31: Di-substituted and multiple group effects Slide 32: Polynuclear aromatics When the number of fused aromatic rings increases, the l for the primary and secondary bands also increase For heteroaromatic systems spectra become complex with the addition of the n p* transition and ring size effects and are unique to each case Slide 33: Rules for calculating absorption maximum for derivatives of benzenes Like Wood word fisher rules, Scott devised a set of rules for calculating the absorption maximum for the derivatives of Acyl benzenes. These rules help in estimating the position of absorption maximum in ethanol in a number of mono substituted aromatic ketones, aldehydes,acid and esters. For the compound of the type 1.The basic value is 246 nm if X is an alkyl group or alicyclic residue. 2. If X is hydrogen atom, the basic value becomes 250nm and 3. The basic value is 230 nm if X is OH and 245nm if X is OR. Slide 34: The structural increments in nm for further substation on the aromatic ring in the ortho meta & para position are given in the table Slide 35: 1.Calculating the absorption maximum in ethanol for P-Chloroacetophenone. In this case X is an alkyl group and thus the basic value is 246nm Basic value -246nm Cl- substitution at para position -10nm Calculated value -256nm Observed value -254nm 2. Calculating the absorption maximum for the following compound Basic value -246nm OH- substation at Para position -25nm OH - substation at meta position -7nm Calculated value -278nm Observed value -281nm Slide 36: APPLICATIONS OF U.V. – VISUAL SPECTROSCOPY QUALITATIVE ANALYSIS Detection of impurities Identification of compound 2. QUANTITATIVE ANALYSIS 3. SRUCTURAL STUDIES Effect of conjugation Effect of cross conjugation Effect of Geometric isomerism Keto-enol tautomerism Effect of alkyl substitution 4. DETERMINATION OF MOLECULAR WEIGHT 5. DETERMINATION OF DISSOCATION CONSTANT OF ACIDS AND BASES 6. CHEMICAL KINETICS 7. QUANTATIVE ANLYSIS OF PHARMACEUTICAL SUBSTANCES Slide 37: 1. QUALITATIVE ANALYSIS Detection of impurities : The impurities present in the sample can be detected from the absorption spectrum by considering the following factors. a) Presence of additional peak in the spectrum Additional peaks can be due to impurities present in the sample and can be compared with that of standard. b) Enhanced the peak intensities c) By measuring the absorbance of the sample at specific wavelength Examples: The presence of phenones (310nm) in Adrenaline (210nm) can be determined . Cyclohexane contains benzene as impurity. Benzene can be detected by measuring the absorbance at specific wavelength of 255 nm. Slide 38: ii. Identification of compound Compound containing lone pair of electrons or conjugated double bonds absorb UV radiation and gives characteristic absorption spectrum. The unknown compound can be identified by comparing its absorption spectrum with that of the standard. UV -Spectroscopy helps in structure elucidation of organic molecules. The presence or absence of unsaturation, the presence of hetero atoms like S,O,N or halogens can be determined. Slide 39: The following figure will show how the presence of peaks corresponds to the unsaturation/saturation and presence/absence of hetero atoms. Slide 40: From the location of peaks, some information about a molecule can be obtained. A combination of peaks is also observed. Thus the results of the spectral studies help in structure elucidation, whether the compound is saturated / unsaturated, hetero atom is present/absent, etc. 2. QUANTITATIVE ANALYSIS Quantitative analysis by UV-visible spectroscopy helps to determine the concentration and amount of drug present in a given sample solution as well as its percentage purity. Quantitative analysis of compound can be done by following methods. 1) Using E1% values This method can be used for estimation of drug from raw materials and formulation. This method is used when reference standard unavailable. 1CM Slide 41: The % purity can be determined by the following formula. Observed Absorbance x 100 E value x Concentration % purity = 2) Reference standard method This method involves measurement of average E1% value. The average E1% value can be determined by measuring the absorbance of different standard solution and calculate their average. 1CM 1CM 3) Direct comparison method this method also called as single standard method. It involves the measurement of absorbance of a standard solution whose concentration is known and sample solution whose concentration is to be determined. Slide 42: Calibration curve method This method also called as multiple standard method. In a single standard method , when error is introduced in preparing the solution or measurement of absorbance. The error in result would be greater. To eliminate or to minimize this error, we can use calibration curve method. Calibration curve is a plot of concentration on X-axis and absorbance of series of standard solution of known concentration on Y- axis. A straight line which may or may not pas through origin is obtained. A straight line must coincide with maximum number of points. This line is called calibration curve. Slide 43: The absorbance of sample solution is measured and plotted on graph Concentration Absorbance X Y x x x x x x Fig:- Calibration curve passing through the origin Slide 44: 5) Derivative spectrophotometric method : In this method, spectral isolation is achieved rather than chromatographic isolation. In a derivative spectrum the change in absorbance with respect to wavelength (vs.) wavelength is recorded. 1st or 2nd derivative spectrum is recorded and the characteristic peak for the individual component can be identified and quantified , using a calibration curve of pure substance. 6) Difference spectrophotometric method: This method is especially useful to quantify a substance when interfering species are present. The principle is that the absorbance difference between two forms of the same drug is measured. The absorbance difference is achieved by using pH manipulation using a pair of buffers. This method is used to quantify drugs in biological fluids. Slide 45: 3. SRUCTURAL STUDIES i. Effect of conjugation Extended conjugation shifts the λmax to longer λ (Bathochromic shift or red shifts). On the other hand reduction of the compound or saturation of double bonds leads to the opposite effect i.e. hypsochromic shifts (blue shifts- λmax shorter λ) . Examples : Slide 46: ii. Effect of cross conjugation Cross conjugation has no effect on λmax which can be shown by the following illustration. Eg. Progesterone has λmax at 241nm and has the structure (1) where as prednisone and prednisolone have structure (2), but the λmax is at 241nm and remains unaffected. Hence cross conjugation has no effect on λmax. (I) Conjugation λmax =241nm (II) Cross Conjugation λmax =241nm Slide 47: iii. Effect of Geometric isomerism: Of the Cis and trans isomers, trans isomers absorbs at longer wavelength than Cis isomer. Ex.-Calciferol (Cis isomer) λmax-265nm Isovit-D2 (all trans) λmax-287nm Hence conversion from cis to trans isomer results in bathochromic shifts and hypsochromic shifts and hypochromic effect. Of the two Stilbenes (C6H5-CH=CH-C6H5) , Slide 48: iv. keto-enol tautomerism: Keto and enol forms of a substance have different uv absorption pattern. Using this principle, the parentage of keto and enol form in a mixture, the presence of enol in ether or alcohols can be calculated. V. Effect of alkyl substitution: Alkyl substitution shifts the λmax to longer λ (bathochromic shifts) . Slide 49: No. of rings : The addition of rings, causes bathochromic shifts. The following compounds will give a brief idea of the effect of alkyl substitution. Slide 50: 4. DETERMINATION OF MOLECULAR WEIGHT Both UV and Visible spectroscopy can be used to determined the molecular weight of the compound. The sample whose molecular weight is to be determined is made to react with suitable reagent . The molar absorptivity (ε) of the sample-reagent complex is similar to that of molar absorptivity (ε) of the reagent. Beer-Lambert’s Low can be used to determined the molecular weight of the sample. A = εCt(Beer-Lambert’s Low) where, A – Absorbance of the sample ε – Molar absorptivity C – Concentration of the sample t – Path length Slide 51: 5. DETERMINATION OF DISSOCATION CONSTANT OF ACIDS AND BASES:- Dissociation constant of acids, bases and acid-base indicator can be determined using UV absorption spectrum. Ex.- methyl orange, methyl red, etc CHEMICAL KINETICS : Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is passed through the reaction cell and the absorbance changes can be followed. Slide 52: 7. QUANTATIVE ANLYSIS OF PHARMACEUTICAL SUBSTANCES ASSAY OF MEDICINAL SUBSTANCE: Many drugs either in the from of a raw material or in the from of formulation can be conveniently assayed by making a suitable solution of drug in a solvent and measuring the absorbance at specific wavelength. Although several drugs can be analyzed by uv spectrophotometric method are as follow. Slide 53: REFERENCES Sharma YR. Elementary organic spectroscopy principles and chemical applications. 1st ed. S. Chand and Company ltd; New Delhi :2008. Chatwal GR, Anand SK. Instrumental methods of chemical analysis. 1st ed. Himalaya Publishing house; Mumbai: 2004. Jag Mohan. Organic spectroscopy principles and applications. 1st ed. Narosa publishing House; New Delhi: 2001. Sharma BK. Instrumental methods of chemical analysis. 24th ed. Goel Publishing house; Meerut: 2005. S. Ravi Shankar. Text book of pharmaceutical analysis. 3rd ed. Rx publication; Tirunelveli: 2006. O.V.K. Reddy. Pharmaceutical analysis. Pulse publication; Hyderabad. Slide 54: THANK YOU You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.
u.v.- visible spectroscopy bhavesh.gv89 Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 2634 Category: Education License: All Rights Reserved Like it (4) Dislike it (0) Added: January 21, 2011 This Presentation is Public Favorites: 3 Presentation Description Solvent effects Woodward-Feiser Rules for Calculating Absorption Maxima Application of U.V.-Visual spectroscopy Comments Posting comment... By: vannathankandi (1 month(s) ago) Sir. this presentation is very helpful to me. Kindly send this at saheedvk@gmail.com Thank you Saving..... Post Reply Close Saving..... Edit Comment Close By: sudheer28 (2 month(s) ago) nice presentation.............. pl. send this presentation at k_sudheer28@yahoo.co.in address Saving..... Post Reply Close Saving..... Edit Comment Close By: navinmodi (8 month(s) ago) sir i want u r presentation for my seminar purpose. i liked u r presentation. Saving..... Post Reply Close Saving..... Edit Comment Close By: gopanna (10 month(s) ago) hello mr. bhavesh i am interested to download your uv ppt , may i download it. Saving..... Post Reply Close Saving..... Edit Comment Close By: hetalkagarana (11 month(s) ago) Hello,this presentation can be very helpful to me,can u provide me copy of this on chhayakagarana@gmail.com?thank you. Saving..... Post Reply Close Saving..... Edit Comment Close loading.... See all Premium member Presentation Transcript Slide 1: PRESENTED BY Bhavesh G. Vaghasiya M.Pharm 1st year Maratha Mandal’s college of pharmacy, Belgaum. UV- VISUAL SPECTROSCOPY Department of Pharmaceutics Bhavesh.gv89@gmail.com Slide 2: CONTENTS Solvent effects Woodward-Feiser Rules for Calculating Absorption Maxima Application of U.V.-Visual spectroscopy Slide 3: SOLVENT EFFECTS A most suitable solvent is one that dose not it self absorb in the region under investigation. A dilute solution of the sample is always prepared for the spectral analysis. Most commonly used solvent is 95% ethanol as it is cheap and is transparent down to 210 nm. Commercial ethanol should not be used because it is having benzene which absorbs strongly in the UV region. Some other solvents which are commonly used are water, methyl alcohol, cyclohexane, acetonitrile, diethyl ether etc. Slide 4: SOLVENTS USED IN UV SPECTROSCOPY Slide 5: Hexane and other hydrocarbons can be used because these are less polar and have least interactions with the molecule. For ultra-violet spectroscopy, ethanol, water and cyclohexane are generally used. The position and the intensity of absorption maximum is shifted for a particular chromophore by changing the polarity of the solvent. The absorption maximum for the non-polar compounds is the same in alcohol(polar) as well as in hexane(non-polar). The absorption maximum for the polar compounds is usually shifted with the change in polarity of the solvent. Slide 6: α,β- unsaturated carbonyl compounds shows two different shifts. n π* transition (less intense) In such case, the absorption band moves to shorter wavelength by increasing the polarity of solvent. Eg: absorption maximum of acetone is said to 279nm in hexane as compared to 264 nm in water. A B C D Non polar Polar solvent CD>AB n π* Slide 7: π π* transition (intense) In such case, the absorption band moves to longer wavelength by increasing the polarity of the solvent. The dipole interaction with the solvent molecules lower the energy of the excited state more than that of the ground state. Thus, the value of absorption maximum in ethanol will be greater than that observed in hexane. A B C D Polar solvent Non polar AB>CD π π* Slide 8: Choice of solvent It should not itself absorb radiation in region under investigation. It should be less polar so that it has minimum interaction with the solute molecules. The most commonly employed solvent is 95% ethanol. it is cheap, has good dissolving power and dose not absorb radiation above 210 nm. In other word it is transparent above 210 nm. Some other solvents which are transparent above 210 nm are n-hexane, cyclohexane, methanol, water and ether. Slide 9: Benzene, chloroform and carbon tetrachloride cannot be used because they absorb in the range of 240-280 nm. Hexane and other hydarocarbon are sometimes preferred to polar solvents because they have minimum interaction with the solute molecules. The absorbance of solvent should be checked before use because the presence of small amounts of impurities may give rise to appreciable absorption in the range of the sample. Slide 10: UV spectra of phenol in ethanol and in isooctane Slide 11: WOODWARD-FEISER RULES FOR CALCULATING ABSORPTION MAXIMA Introduction Different terms Rules for conjugated dienes, trienes and polyenes Rules for α,β unsaturated carbonyl compound Rule for Aromatic compound Contents :- Slide 12: INTRODUCTION From the study of the ultraviolet absorption spectra of a large number of compounds, Woodward (1941) gave certain rules for correlating λ max with molecular structure. Scott-Feiser (1959): modified rule with more experimental data. The modified rules known as Woodward-fieser rules, can be used to calculate the position of λ max for a given structure by relating the position of λ max with the position and degree of substitution of chromophore. longer the conjugated system, greater is the wavelength of absorption a maximum. The intensity of the absorption also increases with the increase in length of the chromophore. Slide 13: Different Terms Alicylic dienes or dienes contained in an open chain system i.e, where basic unit is butadiene system. 2. Homo-annular conjugated double bonds Are the conjugated double double bond present in the same ring. it is also called as Homodiene. some examples of this type are: Slide 14: 3. Hetero-annular conjugated double bonds Are the double bonds which are present in different ring. for example. 4. Endocyclic double bond The double bond present in a ring only (inside). 5. Exocyclic double bond The double bond is a part of the ring (outside). Slide 15: Woodward – Fieser’s rules for conjugated dienes,trienes,polyenes etc. According to this rules each type of dienes or triens system is having a certain fixed value at which absorption takes place. This constitutes the basic value or parent value. This value of absorption maximum depends upon …… (I) The number of alkyl substitution or ring residues (II) The number of double bonds which extending conjugation (III) The presence s of polar groups such as Cl,Br,OR, are added to the basic value to obtain λ max for a particular compound. Slide 16: The parent values and increment for different substituents / groups (A) Parent value :- 1) Acyclic conjugated diens and Hetero annular conjugated diens 215 nm 2) Homo annular conjugated diens 253 nm 3) Acyclic triens 245 nm 4) Butadiene system 217 nm (B) Increment :- 1) Each alkyl substitution or ring residue 5 nm 2) Exocyclic double bond 5 nm 3) Double bond extending conjugation 30 nm (C) Auxochromes :- -OR 6 nm -SR 30 nm -Cl, -Br 5 nm -NR2 60 nm -OCOCH3 6 nm Slide 17: EXAMPLES 1. Calculate absorption maximum in UV Spectrum of 2,4 Hexadiene. Butadiene system; basic value = 217nm 2 Alkyl substituent(2X5nm) = 10nm Calculated value = 227 nm Observed value = 227 nm Slide 18: 2. Calculate max for 1,4- dimethylcyclohex-1,3-diene Parent value for homoannular ring : = 253 nm Two alkyl substituents : 2 x 5 = 10 nm Two ring residue : 2 x 5 = 10 nm calculated value : = 273 nm observed value : = 263 nm H3C H3C CH3 CH3 Slide 19: 3. Calculate max of following compound Parent value for heteroannular diene : = 215 nm Four ring residue : 4 X5 = 20 nm Calculated value : = 235 nm Observed value : = 236 nm Slide 20: POLY-enes AND POLY-ynes With increase in the number of double bonds in conjugation, the values of absorption maximum as well as intensity increase. The values of absorption maximum as well extinction coefficient in case –(CH=CH)n-where n>3 are given below. max ε max i. n = 3 275 nm 30,000 ii. n = 4 310 nm 76,000 iii. n = 5 342 nm 122,000 etc. Slide 21: If the Polynes contain more than four double bonds, then Fieser-kuhn rules are applied. According to this approach, both λ max and Emax are related to number of conjugated double bonds as well as other structural units by following equations. λ max =114+5M+n(48.0-1.7n)-16.5 Rendo – 10 Rexo Emax = (1.74 x 104)n Where n = No. of conjugated double bonds. M = No of alkyl or alkyl like substituents on conjugated system. Rendo = No of rings with endocylic double bonds in conjugated system. Rexo = No of rings with exocylic double bonds. Slide 22: EXAMPLE In case of Lycopene In this compound only eleven bonds are in conjugation. Thus, n =11. In addition to this eight substituents.[methyl groups & chain residues]. Thus M =8. As there is no ring system there are neither exo nor Endocylic double bonds in this conjugated system. λ max is calculated as follow. λ max =114+5(8)+11[48.0-1.7(11)]-0-0 =476nm the observed value is found to be 476nm(hexanes Emax =19.1*104 Slide 23: Woodward – Fieser’s rules for calculating absorption maximum in α,β-unsaturated corbonyl compounds Woodward and fieser framed certain rules for estimating the absorption maximum for α-β unsaturated carbonyl compounds. The rules were later modified by Scott in 1964. α-β unsaturated ketones are characterized by a strong max in the region 230-260 nm in alcohol and a weak max in the region 315-320 nm. The basic value for α-β unsaturated Acyclic or six membered ketone is 215 nm. Slide 24: Parent value :- 1) α , β , unsaturated acyclic or 215 nm six membred ring Kitone 2) α , β , unsaturated 202nm five membred ring Kitone 3) α , β , unsaturated aldehyde 207 nm (B) Increment :- 1) Each alkyl substitution or ring residue 10 nm at α position 12 nm at β position 18 nm at γ position 5 nm 2) Exocyclic double bond 5 nm 3) Double bond extending conjugation 30 nm 4)Homo anuular conjugated dienes 39 nm The parent values and increment for different substituents / groups Slide 25: (C) Auxochromes :- α – position β – position γ – position -OH 35 nm 30 nm 50 nm -OR 35 nm 30 nm 17 nm -SR - 85 nm - -Cl 15 nm 12 nm - -Br 25 nm 30 nm - -NR2 - 95 nm - -O-COCH3 6 nm 6 nm 6 nm Slide 26: In these compounds, the actual spectra obtained are affected considerably by the nature of the solvent employed. Hence a solvent correction is applied to the calculated value to get the value for that particular solvent. For common solvent the following correction should be applied in computing absorption maxima. water 8 nm methanol 0 nm chloroform -1 nm hexane -11 nm cyclohexen -11nm Slide 27: 1.Calculate λ max for given compound 1. α -β unsaturated ketone 215nm 2. 2 β alkyl substituents(2X12nm) 24nm Calculated value 239nm Observed value 237nm c EXAMPLES Slide 28: Parent value for ,-unsaturated 6 = 215 nm membered cyclic ketone One ring residue at position = 10nm Two ring residue at position 2 x 12 = 24 nm Double bond exocyclic to two ring 2 x 5 = 10nm Calculated value = 259nm Observed value = 256nm 2.Calculate λ max for given compound Slide 29: Aromatic Compounds The formulation of empirical rules is for the most part is not efficient (there are more exceptions than rules) If the group attached to the ring bears n electrons, they can induce a shift in the primary and secondary absorption bands Slide 30: Electron-donating and electron-withdrawing effects Electron donating Electron withdrawing Slide 31: Di-substituted and multiple group effects Slide 32: Polynuclear aromatics When the number of fused aromatic rings increases, the l for the primary and secondary bands also increase For heteroaromatic systems spectra become complex with the addition of the n p* transition and ring size effects and are unique to each case Slide 33: Rules for calculating absorption maximum for derivatives of benzenes Like Wood word fisher rules, Scott devised a set of rules for calculating the absorption maximum for the derivatives of Acyl benzenes. These rules help in estimating the position of absorption maximum in ethanol in a number of mono substituted aromatic ketones, aldehydes,acid and esters. For the compound of the type 1.The basic value is 246 nm if X is an alkyl group or alicyclic residue. 2. If X is hydrogen atom, the basic value becomes 250nm and 3. The basic value is 230 nm if X is OH and 245nm if X is OR. Slide 34: The structural increments in nm for further substation on the aromatic ring in the ortho meta & para position are given in the table Slide 35: 1.Calculating the absorption maximum in ethanol for P-Chloroacetophenone. In this case X is an alkyl group and thus the basic value is 246nm Basic value -246nm Cl- substitution at para position -10nm Calculated value -256nm Observed value -254nm 2. Calculating the absorption maximum for the following compound Basic value -246nm OH- substation at Para position -25nm OH - substation at meta position -7nm Calculated value -278nm Observed value -281nm Slide 36: APPLICATIONS OF U.V. – VISUAL SPECTROSCOPY QUALITATIVE ANALYSIS Detection of impurities Identification of compound 2. QUANTITATIVE ANALYSIS 3. SRUCTURAL STUDIES Effect of conjugation Effect of cross conjugation Effect of Geometric isomerism Keto-enol tautomerism Effect of alkyl substitution 4. DETERMINATION OF MOLECULAR WEIGHT 5. DETERMINATION OF DISSOCATION CONSTANT OF ACIDS AND BASES 6. CHEMICAL KINETICS 7. QUANTATIVE ANLYSIS OF PHARMACEUTICAL SUBSTANCES Slide 37: 1. QUALITATIVE ANALYSIS Detection of impurities : The impurities present in the sample can be detected from the absorption spectrum by considering the following factors. a) Presence of additional peak in the spectrum Additional peaks can be due to impurities present in the sample and can be compared with that of standard. b) Enhanced the peak intensities c) By measuring the absorbance of the sample at specific wavelength Examples: The presence of phenones (310nm) in Adrenaline (210nm) can be determined . Cyclohexane contains benzene as impurity. Benzene can be detected by measuring the absorbance at specific wavelength of 255 nm. Slide 38: ii. Identification of compound Compound containing lone pair of electrons or conjugated double bonds absorb UV radiation and gives characteristic absorption spectrum. The unknown compound can be identified by comparing its absorption spectrum with that of the standard. UV -Spectroscopy helps in structure elucidation of organic molecules. The presence or absence of unsaturation, the presence of hetero atoms like S,O,N or halogens can be determined. Slide 39: The following figure will show how the presence of peaks corresponds to the unsaturation/saturation and presence/absence of hetero atoms. Slide 40: From the location of peaks, some information about a molecule can be obtained. A combination of peaks is also observed. Thus the results of the spectral studies help in structure elucidation, whether the compound is saturated / unsaturated, hetero atom is present/absent, etc. 2. QUANTITATIVE ANALYSIS Quantitative analysis by UV-visible spectroscopy helps to determine the concentration and amount of drug present in a given sample solution as well as its percentage purity. Quantitative analysis of compound can be done by following methods. 1) Using E1% values This method can be used for estimation of drug from raw materials and formulation. This method is used when reference standard unavailable. 1CM Slide 41: The % purity can be determined by the following formula. Observed Absorbance x 100 E value x Concentration % purity = 2) Reference standard method This method involves measurement of average E1% value. The average E1% value can be determined by measuring the absorbance of different standard solution and calculate their average. 1CM 1CM 3) Direct comparison method this method also called as single standard method. It involves the measurement of absorbance of a standard solution whose concentration is known and sample solution whose concentration is to be determined. Slide 42: Calibration curve method This method also called as multiple standard method. In a single standard method , when error is introduced in preparing the solution or measurement of absorbance. The error in result would be greater. To eliminate or to minimize this error, we can use calibration curve method. Calibration curve is a plot of concentration on X-axis and absorbance of series of standard solution of known concentration on Y- axis. A straight line which may or may not pas through origin is obtained. A straight line must coincide with maximum number of points. This line is called calibration curve. Slide 43: The absorbance of sample solution is measured and plotted on graph Concentration Absorbance X Y x x x x x x Fig:- Calibration curve passing through the origin Slide 44: 5) Derivative spectrophotometric method : In this method, spectral isolation is achieved rather than chromatographic isolation. In a derivative spectrum the change in absorbance with respect to wavelength (vs.) wavelength is recorded. 1st or 2nd derivative spectrum is recorded and the characteristic peak for the individual component can be identified and quantified , using a calibration curve of pure substance. 6) Difference spectrophotometric method: This method is especially useful to quantify a substance when interfering species are present. The principle is that the absorbance difference between two forms of the same drug is measured. The absorbance difference is achieved by using pH manipulation using a pair of buffers. This method is used to quantify drugs in biological fluids. Slide 45: 3. SRUCTURAL STUDIES i. Effect of conjugation Extended conjugation shifts the λmax to longer λ (Bathochromic shift or red shifts). On the other hand reduction of the compound or saturation of double bonds leads to the opposite effect i.e. hypsochromic shifts (blue shifts- λmax shorter λ) . Examples : Slide 46: ii. Effect of cross conjugation Cross conjugation has no effect on λmax which can be shown by the following illustration. Eg. Progesterone has λmax at 241nm and has the structure (1) where as prednisone and prednisolone have structure (2), but the λmax is at 241nm and remains unaffected. Hence cross conjugation has no effect on λmax. (I) Conjugation λmax =241nm (II) Cross Conjugation λmax =241nm Slide 47: iii. Effect of Geometric isomerism: Of the Cis and trans isomers, trans isomers absorbs at longer wavelength than Cis isomer. Ex.-Calciferol (Cis isomer) λmax-265nm Isovit-D2 (all trans) λmax-287nm Hence conversion from cis to trans isomer results in bathochromic shifts and hypsochromic shifts and hypochromic effect. Of the two Stilbenes (C6H5-CH=CH-C6H5) , Slide 48: iv. keto-enol tautomerism: Keto and enol forms of a substance have different uv absorption pattern. Using this principle, the parentage of keto and enol form in a mixture, the presence of enol in ether or alcohols can be calculated. V. Effect of alkyl substitution: Alkyl substitution shifts the λmax to longer λ (bathochromic shifts) . Slide 49: No. of rings : The addition of rings, causes bathochromic shifts. The following compounds will give a brief idea of the effect of alkyl substitution. Slide 50: 4. DETERMINATION OF MOLECULAR WEIGHT Both UV and Visible spectroscopy can be used to determined the molecular weight of the compound. The sample whose molecular weight is to be determined is made to react with suitable reagent . The molar absorptivity (ε) of the sample-reagent complex is similar to that of molar absorptivity (ε) of the reagent. Beer-Lambert’s Low can be used to determined the molecular weight of the sample. A = εCt(Beer-Lambert’s Low) where, A – Absorbance of the sample ε – Molar absorptivity C – Concentration of the sample t – Path length Slide 51: 5. DETERMINATION OF DISSOCATION CONSTANT OF ACIDS AND BASES:- Dissociation constant of acids, bases and acid-base indicator can be determined using UV absorption spectrum. Ex.- methyl orange, methyl red, etc CHEMICAL KINETICS : Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is passed through the reaction cell and the absorbance changes can be followed. Slide 52: 7. QUANTATIVE ANLYSIS OF PHARMACEUTICAL SUBSTANCES ASSAY OF MEDICINAL SUBSTANCE: Many drugs either in the from of a raw material or in the from of formulation can be conveniently assayed by making a suitable solution of drug in a solvent and measuring the absorbance at specific wavelength. Although several drugs can be analyzed by uv spectrophotometric method are as follow. Slide 53: REFERENCES Sharma YR. Elementary organic spectroscopy principles and chemical applications. 1st ed. S. Chand and Company ltd; New Delhi :2008. Chatwal GR, Anand SK. Instrumental methods of chemical analysis. 1st ed. Himalaya Publishing house; Mumbai: 2004. Jag Mohan. Organic spectroscopy principles and applications. 1st ed. Narosa publishing House; New Delhi: 2001. Sharma BK. Instrumental methods of chemical analysis. 24th ed. Goel Publishing house; Meerut: 2005. S. Ravi Shankar. Text book of pharmaceutical analysis. 3rd ed. Rx publication; Tirunelveli: 2006. O.V.K. Reddy. Pharmaceutical analysis. Pulse publication; Hyderabad. Slide 54: THANK YOU