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  1. 1. : UV-Vis Spectroscopy Dr. Mahwish Qayyum Organic Spectral Analysis 1
  2. 2. Welcome to My Class! Dr.Mahwish Qayyum
  3. 3. Table of contents •introduction •UV & electronic transitions •Usable ranges & observations •Selection rules •Band Structure •Instrumentation & Spectra •Beer-Lambert Law •Application of UV-spec •Conclusion
  4. 4. 513 PHARMACEUTICAL CHEMISTRY-III (Instrumentation-I) Cr. Hr. 03 Topics include: 5th Semester 513 PHARMACEUTICAL CHEMISTRY-III (Instrumentation-I) (Theory) Cr. Hr. 03 Note:- The topics will be taught with special reference to their Pharmaceutical Applications. Theory, Instrumentation and Pharmaceutical Applications of the following Spectroscopic Methods 1. Atomic Absorption and Emission Spectroscopy. 2. Molecular fluorescence spectroscopy. 3. Flame Photometry. 4. I.R. Spectroscopy. 5. Mass Spectroscopy. 6. NMR Spectroscopy. 7. UV/Visible Spectroscopy 515 PHARMACEUTICAL CHEMISTRY-III (Instrumentation-I) (Laboratory) Cr. Hr. 01 NOTE:- Practical of the subject shall be designed from time to time on the basis of the above mentioned theoretical topics and availability of the requirements, e.g. Determination of the Purity and Composition of the unknown drugs by using at least each of the above techniques .
  5. 5. Reference books PHARMACEUTICAL CHEMISTRY-III (Instrumentation-I) 5th ( Semester) Lough W J, High Performance Liquid Chromatography, Blacki Academic Press, New York, 1996. William Kemp,Organic Spectroscopy, Ellsi Horwood, London, 1990. M Aminuddin & Javed Iqbal, Theory and Practice of Chromatography,University Grants Commission, Islamabad-Pakistan (2000). A H Beckett and J B Stennlake, Practical Pharmaceutical Chemistry,Part I and II, the Aulton Press, London. A M Knevel and F E Digangi, Jenkins’s quantitative Pharmaceutical Chemistry, McGraw-Hill Book Company, New York. Braithwaite and F J Smith, Chromatographic Methods, Chapman andHall, London. E Heftmann, Chromatography, Von Nostrond Reinheld Co, New York,1975. Pryde and M J Gilbert, Applications of High Performance Liquid Chromatography, Chapman & Hall, London, 1979. E Stahl, Thin Layer Chromatography, Springer-Verlag, Berlin, 1969. 10. R Hamilton, Introduction to HPLC, P A Sewell, Chapman & Hall, London, 1982. Organic chemistry by M.YOUNIS
  6. 6. Our Classroom Rules 1 No food in the classroom (unless it's cake/chocolate for me) any confusion then refer to rule 2 again 3.Avoid death by powerpoint so no sleep otherwise its going to be sleep forever 4.Avoid commenting about anything in your paper chat coz save your self by not providng documented proofs (personal experience) 5.Only smarter questions are allowed (Watch out ahsan) 6.Theres a difference in a classroom & common room 7. Pleasee don't refresh your gloss or your hair by facing it towards your teacher face assuming it as a mirror (God you really wanna do that !!! DaAA) 2 Teacher is always correct if you find have
  7. 7. We are going to have a wonderful semester of learning! InshAllah sooooo 
  8. 8. Lets start with the name of great Almighty Allah O My Lord! expand my chest for me. And ease my task for me; and loose a knot from my tongue, (That) they may understand my saying. (20:25-2)
  9. 9. Spectroscopy Spectroscopy is a general term referring to the interactions of various types of electromagnetic radiation with matter. Exactly how the radiation interacts with matter is directly dependent on the energy of the radiation.
  10. 10. The molecular spectroscopy is the study of the interaction of electromagnetic waves and matter. The scattering of sun’s rays by raindrops to produce a rainbow and appearance of a colorful spectrum when a narrow beam of sunlight is passed through a triangular glass prism are the simple examples where white light is separated into the visible spectrum of primary colors. This visible light is merely a part of the whole spectrum of electromagnetic radiation, extending from the radio waves to cosmic rays. All these apparently different forms of electromagnetic radiations travel at the same velocity but characteristically differ from each other in terms of frequencies and wavelength
  11. 11. Early days of medicinal chemistry i) 2 major problems Separation of a chemical substance either from mixture/natural source ii) Chemical analysis bothqualitative and quantitative iii) Early days qualitative (sublimation, crystallization, distillation etc) iv) Quantitative (volumetric/gravimetric) v) Electromagnetic radiations can also give information i.e empirically to presence of certain functional groups.so can help in structural and analytical
  12. 12. Electromagnetic radiation: Transmission of energy radiant energy Heat and light energy Quantum mechanics suggests that e.m radiation dual nature Waves Particle like discrete packets energy
  13. 13. UV Spectroscopy I. Introduction A. UV radiation and Electronic Excitations 1. 2. This energy corresponds to EM radiation in the ultraviolet (UV) region, 100-350 nm, and visible (VIS) regions 350-700 nm of the spectrum For comparison, recall the EM spectrum: -rays X-rays UV IR Microwave Radio Visible 3. 4. 5. RAMI VISUALIZES ULTRA XRAYS GA CAR Using IR we observed vibrational transitions with energies of 8-40 kJ/mol at wavelengths of 2500-15,000 nm For purposes of our discussion, we will refer to UV and VIS spectroscopy as UV 13
  14. 14. Spectroscopy Utilises the Absorption and Emission of electromagnetic radiation by atoms Absorption: Low energy electrons absorb energy to move to higher energy level Emission: Excited electrons return to lower energy states 15
  15. 15. Absorption v. Emission Energy is emitted by electrons returningto lower energy levels 3rd Excited States 2nd 1st Energy is absorbed as electrons jump to higher energy levels Ground State 16
  16. 16. Absorption Spectra Sodium 18
  17. 17. The Spectroscopic Techniques are based on the fact that Light absorbed (Absorption) is directly proportional to the Concentration 19 of the absorbing component.
  18. 18. A FLASH BACK !!!!!!!!! 20
  19. 19. UV-Visible Spectroscopy A UV-visible spectrophotometer measures the amount of energy absorbed by a sample. 21
  20. 20. The optics of the light source in UV-visible spectroscopy allow either visible [approx. 400nm (blue end) to 750nm (red end) ] or ultraviolet (below 400nm) to be directed at the sample under analysis. 22
  21. 21. 23
  22. 22. Why are carrots orange? Carrots contain the pigment carotene which absorbs blue light strongly and reflects orange red and so the carrot appears orange. 400nm 500nm BLUE 420nm 600nm GREEN 520 nm 24 700nm Y E L LO W O R A N G E 600nm RED
  23. 23. Carotene  beta-Carotene forms orange to red crystals and occurs in the chromoplasts of plants and in the fatty tissues of plant-eating animals.  Molecular formula: C40H56  Molar Mass 537  Melting point 178 - 179 °C 25
  24. 24. Absorbance is set to 0% or light transmitted using a solvent blank in a cuvet. This compensates for absorbance by the cell container and solvent and ensures that any absorbance registered is solely due to the component under analysis. The sample to be analysed is placed in a cuvet Qualitative analysis is achieved by determining the radiation absorbed by a sample over a range of wavelengths. The results are plotted as a graph of absorbance/transmittance against wavelength, which is called a UV/visible spectrum. 26
  25. 25. The UV- Visible absorption spectrum for carotene in the non-polar solvent, hexane I NT EN S I TY O F A B S O R P T I O N 700 nm 400nm ultra- violet 320nm 27 visible 460nm 540nm infrared
  26. 26. Although the light absorbed is dependent on pathlength through the cell, a usual standard 1cm pathlength is used so that pathlength can effectively be ignored. Quantitative analysis is achieved in a manner similar to colorimetry. The absorption of a sample at a particular wavelength (chosen by adjusting a monochromator) is measured and compared to a calibration graph of the absorptions of a series of standard solutions. What can be analysed? In its quantitative form, UV-visible spectroscopy can be used to detect coloured species in solution eg. bromine , iodine and organic compounds or metal ions that are coloured, or can be converted into a coloured compound. 28
  27. 27.  Molecular orbital is the nonlocalized fields between atoms that are occupied by bonding electrons. (when two atom orbitals combine, either a low-energy bonding molecular orbital or a high energy antibonding molecular orbital results.)  Sigma ( ) orbital The molecular orbital associated with single bonds in organic compounds  Pi ( ) orbital The molecular orbital associated with parallel overlap of atomic P orbital.  n electrons No bonding electrons
  28. 28. UV Spectroscopy I. Introduction B. C. The Spectroscopic Process The difference in energy between molecular bonding, non-bonding and anti-bonding orbitals ranges from 125-650 kJ/mole 1. 2. 3. 4. In UV spectroscopy, the sample is irradiated with the broad spectrum of the UV radiation If a particular electronic transition matches the energy of a certain band of UV, it will be absorbed The remaining UV light passes through the sample and is observed From this residual radiation a spectrum is obtained with “gaps” at these discrete energies – this is called an absorption spectrum 30
  29. 29. UV Spectroscopy I. Introduction C. Observed electronic transitions 1. The lowest energy transition (and most often obs. by UV) is typically that of an electron in the Highest Occupied Molecular Orbital (HOMO) to the Lowest Unoccupied Molecular Orbital (LUMO) 2. 3. For any bond (pair of electrons) in a molecule, the molecular orbitals are a mixture of the two contributing atomic orbitals; for every bonding orbital “created” from this mixing ( , ), there is a corresponding anti-bonding orbital of symmetrically * * higher energy ( , ) The lowest energy occupied orbitals are typically the anti-bonding orbital is of the highest energy likewise, the corresponding -orbitals are of somewhat higher energy, and their complementary anti-bonding orbital somewhat lower in energy than *. 5. Unshared pairs lie at the energy of the original atomic orbital, most often this energy is higher than or (since no bond is formed, there is no benefit in energy) 31
  30. 30. UV Spectroscopy I. Introduction C. Observed electronic transitions 6. Here is a graphical representation Unoccupied levels Energy Atomic orbital n Atomic orbital Occupied levels Molecular orbitals 32
  31. 31. UV Spectroscopy I. Introduction C. Observed electronic transitions 7. From the molecular orbital diagram, there are several possible electronic transitions that can occur, each of a different relative energy: alkanes carbonyls Energy n unsat cpds. n O,N,S, halogens n carbonyls 33
  32. 32. UV Spectroscopy I. Introduction C. Observed electronic transitions 7. Although the UV spectrum extends below 100 nm (high energy), oxygen in the atmosphere is not transparent below 200 nm 8. Special equipment to study vacuum or far UV is required 9. Routine organic UV spectra are typically collected from 200-700 nm 10. This limits the transitions that can be observed: alkanes 150 nm carbonyls 170 nm unsaturated cmpds. 180 nm n O, N, S, halogens 190 nm n carbonyls 300 nm √ - if conjugated! √ 34
  33. 33. UV Spectroscopy I. Introduction D. Selection Rules 1. Not all transitions that are possible are observed 2. 3. 4. For an electron to transition, certain quantum mechanical constraints apply – these are called “selection rules” For example, an electron cannot change its spin quantum number during a transition – these are “forbidden” Other examples include: • the number of electrons that can be excited at one time • symmetry properties of the molecule • symmetry of the electronic states To further complicate matters, “forbidden” transitions are sometimes observed (albeit at low intensity) due to other factors 35
  34. 34. UV Spectroscopy I. Introduction E. Band Structure 1. Unlike IR (or later NMR), where there may be upwards of 5 or more resolvable peaks from which to elucidate structural information, UV tends to give wide, overlapping bands 2. 3. 4. It would seem that since the electronic energy levels of a pure sample of molecules would be quantized, fine, discrete bands would be observed – for atomic spectra, this is the case In molecules, when a bulk sample of molecules is observed, not all bonds (read – pairs of electrons) are in the same vibrational or rotational energy states This effect will impact the wavelength at which a transition is observed – very similar to the effect of H-bonding on the O-H vibrational energy levels in neat samples 36
  35. 35. UV Spectroscopy I. Introduction E. Band Structure 5. When these energy levels are superimposed, the effect can be readily explained – any transition has the possibility of being observed Disassociation R1 - Rn V4 R1 - Rn V3 R1 - Rn V2 V1 R1 - Rn E1 Vo R1 - Rn Disassociation Energy R1 - Rn V4 R1 - Rn V3 R1 - Rn V2 V1 R1 - Rn E0 Vo R1 - Rn 37
  36. 36. UV Spectroscopy Instrumentation and Spectra A. Instrumentation 1. The construction of a traditional UV-VIS spectrometer is very similar to an IR, as similar functions – sample handling, irradiation, detection and output are required 2. Here is a simple schematic that covers most modern UV spectrometers: I0 I 200 detector UV-VIS sources sample log(I0/I) = A monochromator/ beam splitter optics I0 reference II. 700 , nm I0 38
  37. 37. Components of instrumentation:  Sources  Sample Containers  Monochromators  Detectors
  38. 38. Components of instrumentation:  Sources: Agron, Xenon, Deuteriun, or Tungsten lamps  Sample Containers: Quartz, Borosilicate, Plastic  Monochromators: Quarts prisms and all gratings  Detectors: Pohotomultipliers
  39. 39. Sources Deuterium and hydrogen lamps (160 – 375 nm) D2 + Ee → D2* → D’ + D’’ + h Excited deuterium molecule with fixed quantized energy Dissociated into two deuterium atoms with different kinetic energies Ee = ED2* = ED’ + ED’’ + hv Ee is the electrical energy absorbed by the molecule. ED2* is the fixed quantized energy of D2*, ED’ and ED’’ are kinetic energy of the two deuterium atoms.
  40. 40. Sources Tungsten lamps (350-2500 nm) Low limit: 350 nm 1)Low density 2)Glass envelope
  41. 41. General Instrument Designs Single beam Requires a stabilized voltage supply
  42. 42. General Instrument Designs Double Beam: Space resolved Need two detectors
  43. 43. General Instrument Designs Double Beam: Time resolved
  44. 44. Double Beam Instruments 1.Compensate for all but the most short term fluctuation in radiant output of the source 2.Compensate drift in transducer and amplifier 3.Compensate for wide variations in source intensity with wavelength
  45. 45. Multi-channel Design
  46. 46. UV Spectroscopy II. Instrumentation and Spectra A. Instrumentation 3. Two sources are required to scan the entire UV-VIS band: • Deuterium lamp – covers the UV – 200-330 • Tungsten lamp – covers 330-700 4. 5. 6. As with the dispersive IR, the lamps illuminate the entire band of UV or visible light; the monochromator (grating or prism) gradually changes the small bands of radiation sent to the beam splitter The beam splitter sends a separate band to a cell containing the sample solution and a reference solution The detector measures the difference between the transmitted light through the sample (I) vs. the incident light (I0) and sends this information to the recorder 48
  47. 47. UV Spectroscopy Instrumentation and Spectra A. Instrumentation 7. As with dispersive IR, time is required to cover the entire UV-VIS band due to the mechanism of changing wavelengths 8. 9. A recent improvement is the diode-array spectrophotometer - here a prism (dispersion device) breaks apart the full spectrum transmitted through the sample Each individual band of UV is detected by a individual diodes on a silicon wafer simultaneously – the obvious limitation is the size of the diode, so some loss of resolution over traditional instruments is observed Diode array UV-VIS sources sample II. Polychromator – entrance slit and dispersion device 49
  48. 48. UV Spectroscopy II. Instrumentation and Spectra B. Instrumentation – Sample Handling 1. Virtually all UV spectra are recorded solution-phase 2. 3. 4. Cells can be made of plastic, glass or quartz Only quartz is transparent in the full 200-700 nm range; plastic and glass are only suitable for visible spectra Concentration (we will cover shortly) is empirically determined A typical sample cell (commonly called a cuvet): 50
  49. 49. UV Spectroscopy II. Instrumentation and Spectra B. Instrumentation – Sample Handling 5. Solvents must be transparent in the region to be observed; the wavelength where a solvent is no longer transparent is referred to as the cutoff 6. Since spectra are only obtained up to 200 nm, solvents typically only need to lack conjugated systems or carbonyls Common solvents and cutoffs: acetonitrile chloroform240 cyclohexane 1,4-dioxane 95% ethanol n-hexane methanol isooctane water 190 195 215 205 201 205 195 190 51
  50. 50. UV Spectroscopy II. Instrumentation and Spectra B. Instrumentation – Sample Handling 7. Additionally solvents must preserve the fine structure (where it is actually observed in UV!) where possible 8. 9. H-bonding further complicates the effect of vibrational and rotational energy levels on electronic transitions, dipole-dipole interacts less so The more non-polar the solvent, the better (this is not always possible) 52
  51. 51. UV Spectroscopy II. Instrumentation and Spectra C. The Spectrum 1. The x-axis of the spectrum is in wavelength; 200-350 nm for UV, 200-700 for UVVIS determinations 2. Due to the lack of any fine structure, spectra are rarely shown in their raw form, rather, the peak maxima are simply reported as a numerical list of “lamba max” values or max NH2 max = O O 206 nm 252 317 376 53
  52. 52. UV Spectroscopy II. Instrumentation and Spectra C. The Spectrum 1. The y-axis of the spectrum is in absorbance, A 2. 3. From the spectrometers point of view, absorbance is the inverse of transmittance: A = log10 (I0/I) From an experimental point of view, three other considerations must be made: i. a longer path length, l through the sample will cause more UV light to be absorbed – linear effect ii. iii. the greater the concentration, c of the sample, the more UV light will be absorbed – linear effect some electronic transitions are more effective at the absorption of photon than others – molar absorptivity, this may vary by orders of magnitude… 54
  53. 53. UV Spectroscopy II. Instrumentation and Spectra C. The Spectrum 4. These effects are combined into the Beer-Lambert Law: i. ii. iii. cl for most UV spectrometers, l would remain constant (standard cells are typically 1 cm in path length) concentration is typically varied depending on the strength of absorption observed or expected – typically dilute – sub .001 M molar absorptivities vary by4orders of magnitude: • values of 104-106 10 -106 are termed high intensity absorptions • values of 103-104 are termed low intensity absorptions • values of 0 to 103 are the absorptions of forbidden transitions A is unitless, so the units for 5. A= are cm -1 -1 · M and are rarely expressed Since path length and concentration effects can be easily factored out, absorbance simply becomes proportional to , and the y-axis is expressed as directly or as the logarithm of 55
  54. 54. UV Spectroscopy II. Instrumentation and Spectra D. Practical application of UV spectroscopy 1. UV was the first organic spectral method, however, it is rarely used as a primary method for structure determination 2. 3. 4. It is most useful in combination with NMR and IR data to elucidate unique electronic features that may be ambiguous in those methods It can be used to assay (via max and molar absorptivity) the proper irradiation wavelengths for photochemical experiments, or the design of UV resistant paints and coatings The most ubiquitous use of UV is as a detection device for HPLC; since UV is utilized for solution phase samples vs. a reference solvent this is easily incorporated into LC design UV is to HPLC what mass spectrometry (MS) will be to GC 56
  55. 55. UV Spectroscopy III. Chromophores A. Definition 1. Remember the electrons present in organic molecules are involved in covalent bonds or lone pairs of electrons on atoms such as O or N 2. 3. 4. Since similar functional groups will have electrons capable of discrete classes of transitions, the characteristic energy of these energies is more representative of the functional group than the electrons themselves A functional group capable of having characteristic electronic transitions is called a chromophore (color loving) Structural or electronic changes in the chromophore can be quantified and used to predict shifts in the observed electronic transitions 57
  56. 56. UV Spectroscopy III. Chromophores B. Organic Chromophores 1. Alkanes – only posses energy -bonds and no lone pairs of electrons, so only the high  * transition is observed in the far UV This transition is destructive to the molecule, causing cleavage of the -bond C C C C 58
  57. 57. UV Spectroscopy III. Chromophores B. Organic Chromophores 2. Alcohols, ethers, amines and sulfur compounds – in the cases of simple, aliphatic examples of these compounds the n  * is the most often observed transition; like the alkane  * it is most often at shorter than 200 nm Note how this transition occurs from the HOMO to the LUMO C CN N C nN sp C 3 CN C N anitbonding orbital N N 59
  58. 58. UV Spectroscopy III. Chromophores B. Organic Chromophores 3. Alkenes and Alkynes – in the case of isolated examples of these compounds the  * is observed at 175 and 170 nm, respectively Even though this transition is of lower energy than  *, it is still in the far UV – however, the transition energy is sensitive to substitution 60
  59. 59. UV Spectroscopy III. Chromophores B. Organic Chromophores 4. Carbonyls – unsaturated systems incorporating N or O can undergo transitions (~285 nm) in addition to  * n * Despite the fact this transition is forbidden by the selection rules ( = 15), it is the most often observed and studied transition for carbonyls This transition is also sensitive to substituents on the carbonyl Similar to alkenes and alkynes, non-substituted carbonyls undergo the  * transition in the vacuum UV (188 nm, = 900); sensitive to substitution effects 61
  60. 60. UV Spectroscopy III. Chromophores B. Organic Chromophores 4. Carbonyls – n  * transitions (~285 nm);  * (188 nm) O n C O It has been determined from spectral studies, that carbonyl oxygen more approximates sp rather 2 than sp ! O CO transitions omitted for clarity 62
  61. 61. UV Spectroscopy III. Chromophores C. Substituent Effects General – from our brief study of these general chromophores, only the weak n  * transition occurs in the routinely observed UV The attachment of substituent groups (other than H) can shift the energy of the transition Substituents that increase the intensity and often wavelength of an absorption are called auxochromes Common auxochromes include alkyl, hydroxyl, alkoxy and amino groups and the halogens 63
  62. 62. UV Spectroscopy III. Chromophores C. Substituent Effects General – Substituents may have any of four effects on a chromophore i. Bathochromic shift (red shift) – a shift to longer ; lower energy Hypsochromic shift (blue shift) – shift to shorter ; higher energy iii. Hyperchromic effect – an increase in intensity iv. Hypochromic effect – a decrease in intensity Hyperchromic ii. Hypsochromic Bathochromic Hypochromic 200 nm 700 nm 64
  63. 63. UV Spectroscopy III. Chromophores C. Substituent Effects 1. Conjugation – most efficient means of bringing about a bathochromic and hyperchromic shift of an unsaturated chromophore: H2C max nm CH2 175 15,000 217 21,000 258 35,000 465 125,000 -carotene O n *  * 280 189 12 900 O n *  * 280 213 27 7,100 65
  64. 64. UV Spectroscopy III. Chromophores C. Substituent Effects 1. Conjugation – Alkenes The observed shifts from conjugation imply that an increase in conjugation decreases the energy required for electronic excitation From molecular orbital (MO) theory two atomic p orbitals, 1 and 2 from two sp hybrid carbons combine to form two MOs 1 and 2* in ethylene 66 2
  65. 65. UV Spectroscopy III. Chromophores C. Substituent Effects 2. Conjugation – Alkenes When we consider butadiene, we are now mixing 4 p orbitals giving 4 MOs of an energetically symmetrical distribution compared to ethylene E for the HOMO  LUMO transition is reduced 67
  66. 66. UV Spectroscopy III. Chromophores C. Substituent Effects 2. Conjugation – Alkenes Extending this effect out to longer conjugated systems the energy gap becomes progressively smaller: Lower energy = Longer wavelengths Energy ethylene butadiene hexatriene octatetraene 68
  67. 67. UV Spectroscopy III. Chromophores C. Substituent Effects 2. Conjugation – Alkenes Similarly, the lone pairs of electrons on N, O, S, X can extend conjugated systems – auxochromes Here we create 3 MOs – this interaction is not as strong as that of a conjugated system A Energy nA 69
  68. 68. UV Spectroscopy III. Chromophores C. Substituent Effects 2. Conjugation – Alkenes Methyl groups also cause a bathochromic shift, even though they are devoid of or n-electrons This effect is thought to be through what is termed “hyperconjugation” or sigma bond resonance H C C C H H 70
  69. 69. UV Spectroscopy Next time – We will find that the effect of substituent groups can be reliably quantified from empirical observation of known conjugated structures and applied to new systems This quantification is referred to as the Woodward-Fieser Rules which we will apply to three specific chromophores: 1. Conjugated dienes 2. Conjugated dienones 3. Aromatic systems max = 239 nm 71
  70. 70. Reflection and Scattering Losses
  71. 71. LAMBERT-BEER LAW Power of radiation after passing through the solvent A P Psolvent T Psolution P0 log T Power of radiation after passing through the sample solution log P P0 A abc kc a absorptivi ty b pathlength c concentrat ion
  72. 72. Absorption Variables
  73. 73. Beer’s law and mixtures  Each analyte present in the solution absorbs light!  The magnitude of the absorption depends on its  A total = A1+A2+…+An  A total = 1bc1+ 2bc2+…+ nbcn  If 1 = 2 = n then simultaneous determination is impossible  Need n ’s where ’s are different to solve the mixture
  74. 74. Assumptions Ingle and Crouch, Spectrochemical Analysis
  75. 75. Deviations from Beer’s Law Ir ( I0 ( 2 2 ) 1 2 2 ) 1 Successful at low analyte concentrations (0.01M)! High concentrations of other species may also affect
  76. 76. Effects of Stray Light PS T stray light P PS P0 A PS PS P0 log T A log abc PS P0 A P PS kc 100
  77. 77. UV Spectroscopy IV. Structure Determination A. Dienes 1. General Features For acyclic butadiene, two conformers are possible – s-cis and s-trans s-trans s-cis The s-cis conformer is at an overall higher potential energy than the s-trans; therefore the HOMO electrons of the conjugated system have less of a jump to the LUMO – lower energy, longer wavelength 79
  78. 78. UV Spectroscopy IV. Structure Determination A. Dienes 1. General Features Two possible  * transitions can occur for butadiene 175 nm –forb. 217 nm s-trans The 2 and 2 175 nm 253 nm s-cis * 4 transition is not typically observed: • The energy of this transition places it outside the region typically observed – 175 nm • The  2 For the more favorable s-trans conformation, this transition is forbidden * 3 transition is observed as an intense absorption 80 4 *
  79. 79. UV Spectroscopy IV. Structure Determination A. Dienes 1. General Features * The 2  3 transition is observed as an intense absorption ( = 20,000+) based at 217 nm within the observed region of the UV While this band is insensitive to solvent (as would be expected) it is subject to the bathochromic and hyperchromic effects of alkyl substituents as well as further conjugation Consider: max = 217 253 220 227 227 256 263 nm 81
  80. 80. UV Spectroscopy IV. Structure Determination A. Dienes 2. Woodward-Fieser Rules Woodward and the Fiesers performed extensive studies of terpene and steroidal alkenes and noted similar substituents and structural features would predictably lead to an empirical prediction of the wavelength for the lowest energy  * electronic transition This work was distilled by Scott in 1964 into an extensive treatise on the Woodward-Fieser rules in combination with comprehensive tables and examples – (A.I. Scott, Interpretation of the Ultraviolet Spectra of Natural Products, Pergamon, NY, 1964) A more modern interpretation was compiled by Rao in 1975 – (C.N.R. Rao, rd Ultraviolet and Visible Spectroscopy, 3 Ed., Butterworths, London, 1975) 82
  81. 81. UV Spectroscopy IV. Structure Determination A. Dienes 2. Woodward-Fieser Rules - Dienes The rules begin with a base value for max of the chromophore being observed: acyclic butadiene = 217 nm The incremental contribution of substituents is added to this base value from the group tables: Group Increment Extended conjugation +30 Each exo-cyclic C=C +5 Alkyl +5 -OCOCH3 +0 -OR +6 -SR +30 -Cl, -Br +5 -NR2 +60 83
  82. 82. UV Spectroscopy IV. Structure Determination A. Dienes 2. Woodward-Fieser Rules - Dienes For example: Isoprene - acyclic butadiene = one alkyl subs. Experimental value 220 nm Allylidenecyclohexane - acyclic butadiene = one exocyclic C=C + 5 nm 2 alkyl subs. Experimental value 237 nm 217 nm + 5 nm 222 nm 217 nm +10 nm 232 nm 84
  83. 83. UV Spectroscopy IV. Structure Determination A. Dienes 3. Woodward-Fieser Rules – Cyclic Dienes There are two major types of cyclic dienes, with two different base values Heteroannular (transoid): = 5,000 – 15,000 base max = 214 Homoannular (cisoid): = 12,000-28,000 base max = 253 The increment table is the same as for acyclic butadienes with a couple additions: Group Additional homoannular Increment +39 Where both types of diene are present, the one with the longer becomes the base 85
  84. 84. UV Spectroscopy IV. Structure Determination A. Dienes 3. Woodward-Fieser Rules – Cyclic Dienes In the pre-NMR era of organic spectral determination, the power of the method for discerning isomers is readily apparent Consider abietic vs. levopimaric acid: C OH O abietic acid C OH O levopimaric acid 86
  85. 85. UV Spectroscopy IV. Structure Determination A. Dienes 3. Woodward-Fieser Rules – Cyclic Dienes For example: 1,2,3,7,8,8a-hexahydro-8a-methylnaphthalene heteroannular diene = 214 nm 3 alkyl subs. (3 x 5) +15 nm 1 exo C=C + 5 nm 234 nm Experimental value 235 nm 87
  86. 86. UV Spectroscopy IV. Structure Determination A. Dienes 3. Woodward-Fieser Rules – Cyclic Dienes heteroannular diene = 4 alkyl subs. (4 x 5) +20 nm 1 exo C=C C OH O 214 nm + 5 nm 239 nm homoannular diene = 4 alkyl subs. (4 x 5) +20 nm 1 exo C=C C OH O 253 nm + 5 nm 278 nm 88
  87. 87. UV Spectroscopy IV. Structure Determination A. Dienes 3. Woodward-Fieser Rules – Cyclic Dienes Be careful with your assignments – three common errors: R This compound has three exocyclic double bonds; the indicated bond is exocyclic to two rings This is not a heteroannular diene; you would use the base value for an acyclic diene Likewise, this is not a homooannular diene; you would use the base value for an acyclic diene 89
  88. 88. UV Spectroscopy IV. Structure Determination B. Enones 1. General Features Carbonyls, as we have discussed have two primary electronic transitions: Remember, the  * transition is allowed and gives a high , but lies outside the routine range of UV observation n The n  * transition is forbidden and gives a very low e, but can routinely be observed 90
  89. 89. UV Spectroscopy IV. Structure Determination B. Enones 1. General Features For auxochromic substitution on the carbonyl, pronounced hypsochromic shifts are observed for the n  * transition ( max): O H 293 nm CH3 279 O O Cl 235 NH2 214 O This is explained by the inductive withdrawal of electrons by O, N or halogen from the carbonyl carbon – this causes the n-electrons on the carbonyl oxygen to be held more firmly It is important to note this is different from the auxochromic effect on  * which extends conjugation and causes a bathochromic shift 204 O O O OH In most cases, this bathochromic shift is not enough to bring the  * transition into the observed range 204 91
  90. 90. UV Spectroscopy IV. Structure Determination B. Enones 1. General Features Conversely, if the C=O system is conjugated both the n  * and are bathochromically shifted Here, several effects must be noted: i. the effect is more pronounced for ii. iii.  * bands  * if the conjugated chain is long enough, the much higher intensity  * band will overlap and drown out the n  * band the shift of the n  * transition is not as predictable For these reasons, empirical Woodward-Fieser rules for conjugated enones are for the higher intensity, allowed  * transition 92
  91. 91. UV Spectroscopy IV. Structure Determination B. Enones 1. General Features These effects are apparent from the MO diagram for a conjugated enone: n n O O 93
  92. 92. UV Spectroscopy IV. Structure Determination B. Enones 2. Woodward-Fieser Rules - Enones C C C O Group C C C C C O Increment 6-membered ring or acyclic enone Base 215 nm 5-membered ring parent enone Base 202 nm Acyclic dienone Base 245 nm Double bond extending conjugation 30 Alkyl group or ring residue and higher 10, 12, 18 -OH and higher 35, 30, 18 -OR -O(C=O)R 35, 30, 17, 31 6 -Cl 15, 12 -Br 25, 30 -NR2 95 Exocyclic double bond 5 Homocyclic diene component 39 94
  93. 93. UV Spectroscopy IV. Structure Determination B. Enones 2. Woodward-Fieser Rules - Enones Aldehydes, esters and carboxylic acids have different base values than ketones Unsaturated system Base Value Aldehyde With With 208 or alkyl groups or With 220 alkyl groups 230 alkyl groups 242 Acid or ester With With or or alkyl groups 208 alkyl groups Group value – exocyclic Group value – endocyclic or 7 membered ring 217 double bond +5 bond in 5 +5 95
  94. 94. UV Spectroscopy IV. Structure Determination B. Enones 2. Woodward-Fieser Rules - Enones Unlike conjugated alkenes, solvent does have an effect on max These effects are also described by the Woodward-Fieser rules Solvent correction Water Increment +8 Ethanol, methanol 0 Chloroform -1 Dioxane -5 Ether -7 Hydrocarbon -11 96
  95. 95. UV Spectroscopy IV. Structure Determination B. Enones 2. Woodward-Fieser Rules - Enones Some examples – keep in mind these are more complex than dienes cyclic enone = 215 nm 2 x - alkyl subs. (2 x 12) +24 nm O 239 nm Experimental value 238 nm R O cyclic enone = extended conj. -ring residue -ring residue exocyclic double bond 215 nm +30 nm +12 nm +18 nm + 5 nm 280 nm Experimental 280 nm 97
  96. 96. UV Spectroscopy IV. Structure Determination B. Enones 2. Woodward-Fieser Rules - Enones Take home problem – can these two isomers be discerned by UV-spec O O Eremophilone allo-Eremophilone st nd th Problem Set 1: (text) – 1,2,3a,b,c,d,e,f,j, 4, 5, 6 (1 , 2 and 5 pairs), 8a, b, c Problem Set 2: outside problems/key -Tuesday 98
  97. 97. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 1. General Features Although aromatic rings are among the most widely studied and observed chromophores, the absorptions that arise from the various electronic transitions are complex On first inspection, benzene has six -MOs, 3 filled , 3 unfilled * 99
  98. 98. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 1. General Features One would expect there to be four possible HOMO-LUMO observable wavelengths (conjugation)  * transitions at Due to symmetry concerns and selection rules, the actual transition energy states of benzene are illustrated at the right: u 200 nm (forbidden) u u 260 nm (forbidden) 180 nm (allowed) g 100
  99. 99. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 1. General Features The allowed transition ( = 47,000) is not in the routine range of UV obs. at 180 nm, and is referred to as the primary band The forbidden transition ( = 7400) is observed if substituent effects shift it into the obs. region; this is referred to as the second primary band At 260 nm is another forbidden transition ( = 230), referred to as the secondary band. This transition is fleetingly allowed due to the disruption of symmetry by the vibrational energy states, the overlap of which is observed in what is called fine structure 101
  100. 100. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 1. General Features Substitution, auxochromic, conjugation and solvent effects can cause shifts in wavelength and intensity of aromatic systems similar to dienes and enones However, these shifts are difficult to predict – the formulation of empirical rules is for the most part is not efficient (there are more exceptions than rules) There are some general qualitative observations that can be made by classifying substituent groups -- 102
  101. 101. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 2. Substituent Effects a. Substituents with Unshared Electrons • If the group attached to the ring bears n electrons, they can induce a shift in the primary and secondary absorption bands • • Non-bonding electrons extend the -system through resonance – lowering the energy of transition  * More available n-pairs of electrons give greater shifts G G G G 103
  102. 102. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 2. Substituent Effects a. Substituents with Unshared Electrons • The presence of n-electrons gives the possibility of n  • • * transitions If this occurs, the electron now removed from G, becomes an extra electron in the anti-bonding * orbital of the ring This state is referred to as a charge-transfer excited state G * G G G * * * 104
  103. 103. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 2. Substituent Effects a. Substituents with Unshared Electrons • pH can change the nature of the substituent group • deprotonation of oxygen gives more available n-pairs, lowering • transition energy protonation of nitrogen eliminates the n-pair, raising transition energy Primary Substituent max Secondary max -H 203.5 7,400 254 204 -OH 211 6,200 270 1,450 -O- 235 9,400 287 2,600 -NH2 230 8,600 280 1,430 -NH3+ 203 7,500 254 169 -C(O)OH 230 11,600 273 970 -C(O)O- 224 8,700 268 560 105
  104. 104. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 2. Substituent Effects b. Substituents Capable of -conjugation • When the substituent is a -chromophore, it can interact with the benzene -system • • With benzoic acids, this causes an appreciable shift in the primary and secondary bands For the benzoate ion, the effect of extra n-electrons from the anion reduces the effect slightly Primary Substituent max Secondary max -C(O)OH 230 11,600 273 970 -C(O)O- 224 8,700 268 560 106
  105. 105. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 2. Substituent Effects c. Electron-donating and electron-withdrawing effects • No matter what electronic influence a group exerts, the presence shifts the primary absorption band to longer • • Electron-withdrawing groups exert no influence on the position of the secondary absorption band Electron-donating groups increase the absorption band and of the secondary 107
  106. 106. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 2. Substituent Effects c. Electron-donating and electron-withdrawing effects Primary Substituent Secondary max max 7,400 254 204 207 7,000 261 225 -Cl 210 7,400 264 190 -Br 210 7,900 261 192 -OH 211 6,200 270 1,450 -OCH3 217 6,400 269 1,480 -NH2 230 8,600 280 1,430 -CN Electron withdrawing 203.5 -CH3 Electron donating -H 224 13,000 271 1,000 C(O)OH 230 11,600 273 970 -C(O)H 250 11,400 -C(O)CH3 224 9,800 -NO2 269 7,800 108
  107. 107. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 2. Substituent Effects d. Di-substituted and multiple group effects • With di-substituted aromatics, it is necessary to consider both groups • • • If both groups are electron donating or withdrawing, the effect is similar to the effect of the stronger of the two groups as if it were a mono-substituted ring If one group is electron withdrawing and one group electron donating and they are para- to one another, the magnitude of the shift is greater than the sum of both the group effects Consider p-nitroaniline: O H2N N O H2N O N O 109
  108. 108. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 2. Substituent Effects d. Di-substituted and multiple group effects • If the two electonically dissimilar groups are ortho- or meta- to one another, the effect is usually the sum of the two individual effects (meta- no resonance; ortho-steric hind.) • • For the case of substituted benzoyl derivatives, an empirical correlation of structure with observed max has been developed This is slightly less accurate than the Woodward-Fieser rules, but can usually predict within an error of 5 nm O R G 110
  109. 109. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 2. Substituent Effects d. Di-substituted and multiple group effects Parent Chromophore max R = alkyl or ring residue R=H 250 R = OH or O-Alkyl O 246 230 Substituent increment R G m p Alkyl or ring residue 3 3 10 -O-Alkyl, -OH, -O-Ring G o 7 7 25 -O- 11 20 78 -Cl 0 0 10 -Br 2 2 15 -NH2 13 13 58 -NHC(O)CH3 20 20 45 -NHCH3 -N(CH3)2 73 20 20 85 111
  110. 110. UV Spectroscopy IV. Structure Determination C. Aromatic Compounds 2. Substituent Effects d. Polynuclear aromatics • When the number of fused aromatic rings increases, the for the primary and secondary bands also increase • For heteroaromatic systems spectra become complex with the addition of the n  * transition and ring size effects and are unique to each case 112
  111. 111. UV Spectroscopy V. Visible Spectroscopy A. Color 1. General • The portion of the EM spectrum from 400-800 is observable to humans- we (and some other mammals) have the adaptation of seeing color at the expense of greater detail 400 500 600 700 800 , nm Violet 400-420 Indigo 420-440 Blue 440-490 Green 490-570 Yellow 570-585 Orange 585-620 Red 620-780 113
  112. 112. UV Spectroscopy V. Visible Spectroscopy A. Color 1. General • When white (continuum of ) light passes through, or is reflected by a surface, those ls that are absorbed are removed from the transmitted or reflected light respectively • What is “seen” is the complimentary colors (those that are not absorbed) • This is the origin of the “color wheel” 114
  113. 113. UV Spectroscopy V. Visible Spectroscopy A. Color 1. General • Organic compounds that are “colored” are typically those with extensively conjugated systems (typically more than five) • Consider -carotene -carotene, max = 455 nm max is at 455 – in the far blue region of the spectrum – this is absorbed The remaining light has the complementary color of orange 115
  114. 114. UV Spectroscopy V. Visible Spectroscopy A. Color 1. General • Likewise: lycopene, max = 474 nm O H N N H O indigo max for lycopene is at 474 – in the near blue region of the spectrum – this is absorbed, the compliment is now red max for indigo is at 602 – in the orange region of the spectrum – this is absorbed, the compliment is now indigo! 116
  115. 115. UV Spectroscopy V. Visible Spectroscopy A. Color 1. General • One of the most common class of colored organic molecules are the azo dyes: N N EWGs EDGs From our discussion of di-subsituted aromatic chromophores, the effect of opposite groups is greater than the sum of the individual effects – more so on this heavily conjugated system Coincidentally, it is necessary for these to be opposite for the original synthetic preparation! 117
  116. 116. UV Spectroscopy V. Visible Spectroscopy A. Color 1. General • These materials are some of the more familiar colors of our “environment” NO2 HO N N H2N OH N N O3S N N NH2 SO3 Para Red Fast Brown Sunset Yellow (Food Yellow 3) 118
  117. 117. The colors of M&M’s Bright Blue Royal Blue Common Food Uses Beverages, dairy products, powders, jellies, confections, condiments, icing. Common Food Uses Baked goods, cereals, snack foods, ice-cream, confections, cherries. Orange-red Lemon-yellow Common Food Uses Gelatins, puddings, dairy products, confections, beverages, condiments. Common Food Uses Custards, beverages, ice-cream, confections, preserves, cereals. Orange Common Food Uses Cereals, baked goods, snack foods, ice-cream, beverages, dessert powders, confections 119
  118. 118. UV Spectroscopy V. Visible Spectroscopy A. Color 1. General • In the biological sciences these compounds are used as dyes to selectively stain different tissues or cell structures • Biebrich Scarlet - Used with picric acid/aniline blue for staining collagen, recticulum, muscle, and plasma. Luna's method for erythrocytes & eosinophil granules. Guard's method for sex chromatin and nuclear chromatin. HO O3S N N N N SO3 120
  119. 119. UV Spectroscopy V. Visible Spectroscopy A. Color 1. General • In the chemical sciences these are the acid-base indicators used for the various pH ranges: • Remember the effects of pH on aromatic substituents Methyl Orange O3S N N Yellow, pH > 4.4 CH3 N CH3 O3S H N N CH3 N CH3 Red, pH < 3.2 121
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