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Edit Comment Close Premium member Presentation Transcript Slide 1: UV VISIBLE SPECTROSCOPY UV & electronic transitions Usable ranges & observations Band Structure Instrumentation & Spectra Beer-Lambert Law Application of UV-spec Calibration BY NANDESH V. PINGALE. Electromagnetic Radiation : Electromagnetic Radiation Radiation is absorbed & emmited in photons. The defining characterstic of a photon is that its energy cannot be split into smaller pieces. Each photon’s energy is defined by its frequency () or wave lenth () or wavenumber (wn) Ephoton = h = hc/ = hc (wn) Two constants appear in these formulas h = plank’s constant, 6.63 x 10-34 J s c = speed of light, 3.00 x 108 m s-1 Wave number (wn) = 1/ Slide 3: UV Spectroscopy Introduction UV radiation and Electronic Excitations The difference in energy between molecular bonding, non-bonding and anti-bonding orbitals ranges from 125-650 kJ/mole 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: 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 UV X-rays IR g-rays Radio Microwave Visible Slide 4: UV Spectroscopy Introduction B. Observed electronic transitions 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) 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 (s, p), there is a corresponding anti-bonding orbital of symmetrically higher energy (s*, p*) The lowest energy occupied orbitals are typically the s; likewise, the corresponding anti-bonding s* orbital is of the highest energy p-orbitals are of somewhat higher energy, and their complementary anti-bonding orbital somewhat lower in energy than s*. Unshared pairs lie at the energy of the original atomic orbital, most often this energy is higher than p or s (since no bond is formed, there is no benefit in energy) Slide 5: UV Spectroscopy Introduction B. Observed electronic transitions Here is a graphical representation Energy s* p s p* n Atomic orbital Atomic orbital Molecular orbitals Occupied levels Unoccupied levels Slide 6: UV Spectroscopy Introduction B. Observed electronic transitions From the molecular orbital diagram, there are several possible electronic transitions that can occur, each of a different relative energy: Energy s* p s p* n s s p n n s* p* p* s* p* alkanes carbonyls unsaturated cmpds. O, N, S, halogens carbonyls Slide 7: UV Spectroscopy Introduction B. Observed electronic transitions Although the UV spectrum extends below 100 nm (high energy), oxygen in the atmosphere is not transparent below 200 nm Special equipment to study vacuum or far UV is required Routine organic UV spectra are typically collected from 200-700 nm This limits the transitions that can be observed: s s p n n s* p* p* s* p* alkanes carbonyls unsaturated cmpds. O, N, S, halogens carbonyls 150 nm 170 nm 180 nm √ - if conjugated! 190 nm 300 nm √ Slide 8: UV Spectroscopy Introduction Band Structure 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 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 Slide 9: UV Spectroscopy Instrumentation and Spectra Instrumentation 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 Here is a simple schematic that covers most modern UV spectrometers: sample reference detector I0 I0 I0 I log(I0/I) = A 200 700 l, nm monochromator/ beam splitter optics UV-VIS sources Slide 10: UV Spectroscopy Instrumentation and Spectra Instrumentation Two sources are required to scan the entire UV-VIS band: Deuterium lamp – covers the UV – 200-330 Tungsten lamp – covers 330-700 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 Slide 11: UV Spectroscopy Instrumentation and Spectra Instrumentation As with dispersive IR, time is required to cover the entire UV-VIS band due to the mechanism of changing wavelengths 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 sample Polychromator – entrance slit and dispersion device UV-VIS sources Diode array Slide 12: UV Spectroscopy Instrumentation and Spectra Instrumentation – Sample Handling Virtually all UV spectra are recorded solution-phase 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): Slide 13: UV Spectroscopy Instrumentation and Spectra The Spectrum The x-axis of the spectrum is in wavelength; 200-350 nm for UV, 200-700 for UV-VIS determinations 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 lmax lmax = 206 nm 252 317 376 Slide 14: UV Spectroscopy Instrumentation and Spectra The Spectrum The y-axis of the spectrum is in absorbance, A 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: a longer path length, l through the sample will cause more UV light to be absorbed – linear effect 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, e this may vary by orders of magnitude… Slide 15: UV Spectroscopy Instrumentation and Spectra The Spectrum These effects are combined into the Beer-Lambert Law: A = e c l 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 by orders of magnitude: values of 104-106 104-106 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 e are cm-1 · M-1 and are rarely expressed Since path length and concentration effects can be easily factored out, absorbance simply becomes proportional to e, and the y-axis is expressed as e directly or as the logarithm of e Slide 16: UV Spectroscopy Instrumentation and Spectra Practical application of UV spectroscopy UV was the first organic spectral method, however, it is rarely used as a primary method for structure determination 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 lmax 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 Slide 17: UV Spectroscopy Chromophores Definition Remember the electrons present in organic molecules are involved in covalent bonds or lone pairs of electrons on atoms such as O or N 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 Slide 18: UV Spectroscopy Chromophores Organic Chromophores Alkanes – only posses s-bonds and no lone pairs of electrons, so only the high energy s s* transition is observed in the far UV This transition is destructive to the molecule, causing cleavage of the s-bond s* s Slide 19: UV Spectroscopy Chromophores Organic Chromophores Alkenes and Alkynes – in the case of isolated examples of these compounds the p p* is observed at 175 and 170 nm, respectively Even though this transition is of lower energy than s s*, it is still in the far UV – however, the transition energy is sensitive to substitution p* p Slide 20: UV Spectroscopy Chromophores Organic Chromophores Carbonyls – unsaturated systems incorporating N or O can undergo n p* transitions (~285 nm) in addition to p p* Despite the fact this transition is forbidden by the selection rules (e = 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 p p* transition in the vacuum UV (188 nm, e = 900); sensitive to substitution effects Slide 21: UV Spectroscopy Chromophores Organic Chromophores Carbonyls – n p* transitions (~285 nm); p p* (188 nm) p p* n sCO transitions omitted for clarity It has been determined from spectral studies, that carbonyl oxygen more approximates sp rather than sp2 ! Slide 22: UV Spectroscopy Chromophores Substituent Effects General – from our brief study of these general chromophores, only the weak n p* 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 Slide 23: UV Spectroscopy Chromophores Substituent Effects General – Substituents may have any of four effects on a chromophore Bathochromic shift (red shift) – a shift to longer l; lower energy Hypsochromic shift (blue shift) – shift to shorter l; higher energy Hyperchromic effect – an increase in intensity Hypochromic effect – a decrease in intensity 200 nm 700 nm e Hypochromic Hypsochromic Hyperchromic Bathochromic Slide 24: UV Spectroscopy Chromophores Substituent Effects Conjugation – most efficient means of bringing about a bathochromic and hyperchromic shift of an unsaturated chromophore: lmax nm e 175 15,000 217 21,000 258 35,000 n p* 280 27 p p* 213 7,100 465 125,000 n p* 280 12 p p* 189 900 Slide 25: UV Spectroscopy Structure Determination Aromatic Compounds 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 p-MOs, 3 filled p, 3 unfilled p* p4* p5* p6* p2 p1 p3 Slide 26: UV Spectroscopy Structure Determination Aromatic Compounds General Features One would expect there to be four possible HOMO-LUMO p p* transitions at observable wavelengths (conjugation) Due to symmetry concerns and selection rules, the actual transition energy states of benzene are illustrated at the right: p4* p5* p6* p2 p1 p3 A1g B2u B1u E1u 260 nm (forbidden) 200 nm (forbidden) 180 nm (allowed) Slide 27: UV Spectroscopy Structure Determination Aromatic Compounds General Features The allowed transition (e = 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 (e = 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 (e = 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 Slide 28: UV Spectroscopy Structure Determination Aromatic Compounds 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 -- Slide 29: UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects 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 p-system through resonance – lowering the energy of transition p p* More available n-pairs of electrons give greater shifts Slide 30: UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Substituents with Unshared Electrons The presence of n-electrons gives the possibility of n p* transitions If this occurs, the electron now removed from G, becomes an extra electron in the anti-bonding p* orbital of the ring This state is referred to as a charge-transfer excited state Slide 31: UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects 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: Slide 32: UV Spectroscopy Visible Spectroscopy Color 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 400 500 600 800 700 Slide 33: UV Spectroscopy Visible Spectroscopy Color General When white (continuum of l) 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” Slide 34: UV Spectroscopy Visible Spectroscopy Color General Organic compounds that are “colored” are typically those with extensively conjugated systems (typically more than five) Consider b-carotene lmax is at 455 – in the far blue region of the spectrum – this is absorbed The remaining light has the complementary color of orange Slide 35: UV Spectroscopy Visible Spectroscopy Color General These materials are some of the more familiar colors of our “environment” Slide 36: UV Spectroscopy Visible Spectroscopy Color 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 Slide 37: ANY QUESTIONS ? 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