Molecular Spectroscopy UV-VIS

Principles

Molecular Spectroscopy Molecular Spectroscopy: the interaction of electromagnetic radiation (light) with matter (organic compounds). This interaction gives specific structural information. Methods of structure determination • Ultraviolet-visible (UV-Vis) Spectroscopy • Nuclear Magnetic Resonances (NMR) Spectroscopy • Infrared (IR) Spectroscopy • Mass (MS) spectrometry (not really spectroscopy)

Principles of molecular spectroscopy: Electromagnetic radiation organic molecule (ground state) light h organic molecule (excited state) relaxation organic molecule (ground state) + h Electromagnetic radiation has the properties of a particle (photon) and a wave. = distance of one wave = frequency: waves per unit time (sec-1, Hz) c = speed of light (3.0 x 108 m • sec-1) h = Plank’s constant (6.63 x 10-34 J • sec)

Electronic Spectroscopy Spectroscopy of the electrons surrounding an atom or a molecule: electron energy-level transitions Atoms: electrons are in hydrogen-like orbitals (s, p, d, f) Molecules: electrons are in molecular orbitals (HOMO, LUMO, …) From http://education.jlab.org (The Bohr model for nitrogen) (The LUMO of benzene)

A molecule absorbs electromagnetic radiation when Principles of molecular spectroscopy: Quantized Energy Levels molecules have discrete energy levels (no continuum between levels) A molecule absorbs electromagnetic radiation when the energy of photon corresponds to the difference in energy between two states

UV-Vis: valence electron transitions organic molecule (ground state) light h organic molecule (excited state) relaxation organic molecule (ground state) + h UV-Vis: valence electron transitions - gives information about p-bonds and conjugated systems Infrared: molecular vibrations (stretches, bends) - identify functional groups Radiowaves: nuclear spin in a magnetic field (NMR) - gives a map of the H and C framework

Molecular UV-Vis Spectroscopy Molecular energy levels and absorbance wavelength:   * and   * transitions: high-energy, accessible in vacuum UV (max <150 nm). Not usually observed in molecular UV-Vis. n  * and   * transitions: non-bonding electrons (lone pairs), wavelength (max) in the 150-250 nm region. n  * and   * transitions: most common transitions observed in organic molecular UV-Vis, observed in compounds with lone pairs and multiple bonds with max = 200-600 nm. Other notes: for n  * transitions, peaks are generally shifted to a lower wavelength with the use of more polar solvents, which interact (hydrogen bond) with the lone pair to lower its energy and make a larger energy gap between it and the * excited state. See Skoog et al., pages 330-335, for more details on this and other effects. Figure from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm

Molecular UV-Vis Spectroscopy: Transitions Major classes of electron transitions HOMO: highest occupied molecular orbital LUMO: lowest unoccupied molecular orbital Types of electron transitions: (1) ,  and n electrons (mostly organics) (2) d and f electrons (inorganics/organometallics) (3) charge-transfer (CT) electrons

,  and n electrons transition

p-molecular orbitals of butadiene 4: 3 Nodes 0 bonding interactions 3 antibonding interactions ANTIBONDING MO 3: 2 Nodes 1 bonding interactions 2 antibonding interactions ANTIBONDING MO 2: 1 Nodes 2 bonding interactions 1 antibonding interactions BONDING MO 1: 0 Nodes 3 bonding interactions 0 antibonding interactions BONDING MO y2 is the Highest Occupied Molecular Orbital (HOMO) y3 is the Lowest Unoccupied Molecular Orbital (LUMO)

UV-Vis light causes electrons in lower energy molecular orbitals to be promoted to higher energy molecular orbitals. HOMO LUMO Chromophore: light absorbing portion of a molecule

Molecular orbitals of conjugated polyenes

Molecules with extended conjugation move toward the visible region Color of absorbed light Color observed l violet 400 nm yellow blue 450 orange blue-green 500 red yellow-green 530 red-violet yellow 550 violet orange 600 blue-green red 700 green

Many natural pigments have conjugated systems Chlorophyll anthocyanin -carotene lycopene

-carotene

Reichardt’s Dyes = solvatochromic dyes

d-f orbital charge-transfer (CT) electrons transition

Molecular UV-Vis Spectroscopy and Transition Metal and Lanthanide/Actinide Complexes d/f orbitals UV-Vis spectra of lanthanides/actinides are particularly sharp, due to screening of the 4f and 5f orbitals by lower shells. Can measure ligand field strength, and transitions between d-orbitals made non-equivalent by the formation of a complex Charge transfer (CT) – occurs when electron-donor and electron-acceptor properties are in the same complex – electron transfer occurs as an “excitation step” MLCT (metal-to-ligand charge transfer) LMCT (ligand-to-metal charge transfer) Ex: tri(bipyridyl)iron(II), which is red – an electron is exicted from the d-orbital of the metal into a * orbital on the ligand ligand field strength

iii) Charge Transfer Complexes ‚ Important analytically because of large e (> 10,000) ‚ Absorption of radiation involves transfer of e- from the donor to orbital associated with acceptor - excited state is product of pseudo oxidation/reduction process ‚ Many inorganic complexes of electron donor (usually organic) & electron acceptor (usually metal) - examples: Iron III thiocyanate Iron II phenanthroline (deep red color) (colorless)

Quantitative aspect

Quantitative Analysis (Beer’s Law): Widely used for Quantitative Analysis Characterization wide range of applications (organic & inorganic) limit of detection  10-4 to 10-5 M (10-6 to 10-7M; current) moderate to high selectivity typical accuracy of 1-3% ( can be ~0.1%) easy to perform, cheap 2) Strategies absorbing species detect both organic and inorganic compounds containing any of these species

Chromophore: light absorbing portion of a molecule Beer’s Law: There is a linear relationship between absorbance and concentration A =  c l A = absorbance c = concentration (M, mol/L) l = sample path length (cm)  = molar absorptivity (extinction coefficient) a proportionality constant for a specific absorbance of a substance

Molecular UV-Vis Spectroscopy: Absorption max is the wavelength(s) of maximum absorption (i.e. the peak position) The strength of a UV-Visible absorption is given by the molar absorption coefficient ():  = 8.7 x 1019 P a where P is the transition probability (0 to 1) – governed by selection rules and orbital overlap, and a is the chromophore area in cm2 Molar absorption coefficient () then gives A via the Beer-Lambert Law: A = ebc

Quantitative UV-Visible Spectroscopy UV-visible spectra can be used for direct quantitative analysis with appropriate calibration

b) non- absorbing species - react with reagent that forms colored product - can also use for absorbing species to lower limit of detection - items to consider: l, pH, temperature, ionic strength - prepare standard curve (match standards and samples as much possible) reagent (colorless) Complex (red) Non-absorbing Species (colorless) As Fe3+ continues to bind protein red color and absorbance increases. When all the protein is bound to Fe3+, no further increase in absorbance.

Standard Addition Method (spiking the sample) - used for analytes in a complex matrix where interferences in the UV/Vis for the analyte will occur: i.e. blood, sediment, human serum, etc.. - Method: (1) Prepare several identical aliquots, Vx, of the unknown sample. (2) Add a variable volume, Vs, of a standard solution of known concentration, cs, to each unknown aliquot. Note: This method assumes a linear relationship between instrument response and sample concentration.

(3) Dilute each solution to an equal volume, Vt. (4) Make instrumental measurements of each sample to get an instrument response, IR.

S = signal or instrument response k = proportionality constant (5) Calculate unknown concentration, cx, from the following equation. Note: assumes a linear relationship between instrument response and sample concentration. Where: S = signal or instrument response k = proportionality constant Vs = volume of standard added cs = concentration of the standard Vx = volume of the sample aliquot cx = concentration of the sample Vt = total volume of diluted solutions

c) Analysis of Mixtures - use two different l’s with different e’s A1 = e1MbcM + e1NbcN (l1) A2 = e2MbcM + e2NbcN (l2) Note: need to solve simultaneous equations

d) Photometric titration - can measure titration with UV-vis spectroscopy. - requires the analyte (A), titrant (T) or titration product (P) absorbs radiation

Example 7: Given: Calculate the concentrations of A and B in solutions that yielded an absorbance of 0.439 at 475 nm and 1.025 at 700 nm in a 2.50-cm cell. Absorbance (1.00 cm cell) Species 475 nm 700 nm A (7.50x10-5 M) 0.155 0.755 B (4.25x10-5 M) 0.702 0.091

instrumentation

Molecular UV-Visible Spectrophotometers The traditional UV-Vis design: double-beam grating systems Sources: Almost universal continuum UV-Vis source is the 2H lamp. Tungsten lamps used for longer (visible) wavelengths. See pg. 313 of Skoog et al. for UV-Vis excitation sources (lamps). Hamamatsu L2D2 lamps Figure from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv1

Molecular UV-Visible Spectrophotometers Diode array detectors can acquire all UV-Visible wavelengths at once. Advantages: Sensitivity (multiplex) Speed Disadvantages: Resolution DAD’s have a higher sensitivity for a given acquisition time, but lower resolution. Figure from Skoog, et al., Chapter 13

Spring 2014 Y4* Y2* Y3* Y2 Y1 p Y1 UV Spectroscopy CHEM 430

log(I0/I) = A I0 I sample detector I0 I0 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: log(I0/I) = A I0 I UV-VIS sources sample 200 700 l, nm detector monochromator/ beam splitter optics I0 I0 reference

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

sample 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 Diode array UV-VIS sources sample Polychromator – entrance slit and dispersion device

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):

UV Spectroscopy Instrumentation and Spectra Instrumentation – Sample Handling 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 Since spectra are only obtained up to 200 nm, solvents typically only need to lack conjugated p systems or carbonyls Common solvents and cutoffs: acetonitrile 190 chloroform 240 cyclohexane 195 1,4-dioxane 215 95% ethanol 205 n-hexane 201 methanol 205 isooctane 195 water 190

UV Spectroscopy Instrumentation and Spectra Instrumentation – Sample Handling Additionally solvents must preserve the fine structure (where it is actually observed in UV!) where possible 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)

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

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…

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 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

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

Qantitative analysis applications

Analyte Method λ (nm) Trace Metals aluminum react with Eriochrome cyanide R dye at pH6; forms red to pink complex 535 arsenic reduce to AsH3 using Zn and react with silver diethyldithiocarbamate; forms red complex cadmium extract into CHCl3 containing dithizone from a sample made basic with NaOH; forms pink to red complex 518 chromium oxidize to Cr(VI) and react with diphenylcarbazide; forms red-violet product 540 copper react with neocuprine in neutral to slightly acid solution and extract into CHCl3/CH3OH; forms yellow complex 457 iron reduce to Fe2+ and react with o-phenanthroline; forms orange-red complex 510 lead extract into CHCl3 containing dithizone from sample made basic with NH3/NH4+ buffer; forms cherry red complex manganese oxidize to MnO4– with persulfate; forms purple solution 525 mercury extract into CHCl3 containing dithizone from acidic sample; forms orange complex 492 zinc react with zincon at pH 9; forms blue complex 620

Inorganic Nonmetals ammonia reaction with hypochlorite and phenol using a manganous salt catalyst; forms blue indophenol as product 630 cyanide react with chloroamine-T to form CNCl and then with a pyridine-barbituric acid; forms a red-blue dye 578 fluoride react with red Zr-SPADNS lake; formation of ZrF62– decreases color of the red lake 570 chlorine (residual) react with leuco crystal violet; forms blue product 592 nitrate react with Cd to form NO2– and then react with sulfanilamide and N-(1-napthyl)-ethylenediamine; forms red azo dye 543 phosphate react with ammonium molybdate and then reduce with SnCl2; forms molybdenum blue 690

Organics phenol react with 4-aminoantipyrine and K3Fe(CN)6; forms yellow antipyrine dye 460 anionic surfactant react with cationic methylene blue dye and extract into CHCl3; forms blue ion pair 652

neocuproine silver diethyldithiocarbamate Eriochrome cyanide R dye

Zincon

Analysis of Glucose Measure the concentration of glucose by detecting the reducing end of the monosaccharide. This group converts the oxidized form of 3,5-dinitrosalicylic acid, DNS, to reduced form which absorbs at 540nm. Amount of reduced DNS proportional to amount of glucose.

Analysis of Glucose

Fe-Phenanthroline

Cd2+ + Dithizon

UV-vis absorption spectrum of (a) dithizone, (b) complex of dithizone with Cd(II) before adsorption, and (c) complex of dithizone with Cd(II) after adsorption

Analysis of Nitrite

Analysis of Surfactants

Organic UV-Vis Spectroscopy (optional)

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

Interpretation of Molecular UV-Visible Spectra UV-Visible spectra can be interpreted to help determine molecular structure, but this is presently confined to the analysis of electron behavior in known compounds. Information from other techniques (NMR, MS, IR) is usually far more useful for structural analysis However, UV-Vis evidence should not be ignored! UV-vis spectra are useful but do not have the information content of other spectroscopic techniques. Typical UV-Visible spectra of small organic molecules are shown here, from Skoog et al. Figure 14-4. Note the lambda(max) and differences in molar absorptivity for each of the spectra. Sometimes compounds in non-viscous solvents (and especially in the gas phase) will show fine structure in the UV-Visible spectrum caused by vibrational energy levels splitting the electronic transitions. Figure from Skoog, et al., Chapter 14

Calculation of Molar Absorption Coefficient The molar absorption coefficient () for each absorbance in a UV spectrum is calculated as follows: , Molar Abs Coeff (AU mol-1 cm-1) = A x mwt / mass x pathlength Solvent “cutoffs” for UV-visible work: Solvent UV Cutoff (nm) Acetonitrile (UV grade) 190 Acetone 330 Dimethylsulfoxide 268 Chloroform (1% ethanol) 245 Heptane 200 Hexane (UV grade) 195 Methanol 205 2-Propanol Tetrahydrofuran (UV grade) 212 Water Molar abosrption coefficient is used to assess the “response factor” of a particular UV-Vis detectable analyte. See page 342 of Skoog et al. for additional info about solvents used in UV-Vis spectrometry. Burdick and Jackson High Purity Solvent Guide, 1990

Interpretation of UV-Visible Spectra Although UV-Visible spectra are no longer frequently used for structural analysis, it is helpful to be aware of well-developed interpretive rules. Examples: Woodward-Fieser rules for max dienes and polyenes Extended Woodward rules for unsaturated ketones Substituted benzenes (max base value = 203.5 nm) Substituent (X) Increment (nm) -CH3 3.0 -Cl 6.0 -OH 7.0 -NH2 26.5 -CHO 46.0 -NO2 65.0 There are several well-developed (and famous) rules for the interpretation of UV-Visible spectra, which were derived by synthesizing a variety of related compounds, recording their spectra, and determining trends in the results, usually in conjunction with Huckel or Extended Huckel Theory. It is worth remembering these rules so that a given UV-Visible spectrum can be checked for consistency. It is especially useful to know these rules in HPLC analysis, to help select wavelengths for UV detection of chromatographic peaks. See E. Pretsch, et al., Structure Determination of Organic Compounds, Springer, 2000. (Chapter 8).

Interpretation of UV-Visible Spectra Other examples: The conjugation of a lone pair on a enamine shifts the max from 190 nm (isolated alkene) to 230 nm. The nitrogen has an auxochromic effect. Why does increasing conjugation cause bathochromic shifts (to longer wavelengths)? The effects of conjugation are distinct – increasing conjugation shifts the UV absorbance to longer wavelengths (lower energies). Fine structure caused by coupling of vibrational and electronic states can also be observed in the UV spectra of highly conjugated compounds. See E. Pretsch, et al., Structure Determination of Organic Compounds, Springer, 2000. (Chapter 8). Figures from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm

Application of UV/Vis Spectroscopy Common Problems: Mixtures: blank samples often contain multiple absorbing species. the absorbance is the sum of all the individual absorbencies A= A1 + A2 +A3 + … = e1bc + e2bc + e3bc … substances in both the blank and sample which absorb can be “blanked out” in both double and single beam spectrometers.

Molar Absorptivities (e) in UV-Vis Range: e = 8.7 x 1019 PA Applications: Molar Absorptivities (e) in UV-Vis Range: e = 8.7 x 1019 PA P – transition probability (ranges from 0.1 to 1, for likely transitions) A – cross-section area of target molecule (cm2) - ~10-15 cm2 for typical organics - emax = 104 to 105 L/mol-cm - e < 103 – low intensity (P #0.01) Name Structure lmax e but-1-en-3-yne 219 7,600 cyclohex-2-enone 225 10,300 toluene 206 7,000 3,4-dimethylpent-3-en-2-one 246 5,300

- For Compounds with Multiple Chromophores: ‚ If greater then one single bond apart - e are additive - l constant CH3CH2CH2CH=CH2 lmax= 184 emax = ~10,000 CH2=CHCH2CH2CH=CH2 lmax=185 emax = ~20,000 ‚ If conjugated - shifts to higher l’s (red shift) H2C=CHCH=CH2 lmax=217 emax = ~21,000

Example 6: The equilibrium constant for the conjugate acid-base pair HIn + H2O H3O+ + In- Calculate the absorbance at 430 nm for an indicator concentration of 3.00x10-4 M K = 8.00x10-5 e = 8.04x103 e = 0.755x103

B) Absorbing Species in UV/Vis: Applications: B) Absorbing Species in UV/Vis: 1) Electronic transitions involving organic compounds, inorganic compounds, complexes, etc. Basic process: M + hn  M* 10-8 – 10-9s M*  M + heat (or fluorescence, light, or phosphorescence) or M*  N (new species, photochemical reaction) Note: excited state (M*) is generally short and heat produced not generally measurable. Thus, get minimal disturbance of systems (assuming no photochemical reaction)

2) Absorption occurs with bonding electrons. E(l) required differs with type of bonding electron. - UV-Vis absorption gives some information on bonding electrons (functional groups in a compound. Most organic spectra are complex ‚ electronic and vibration transitions superimposed absorption bands usually broad ‚ detailed theoretical analysis not possible, but semi-quantitative or qualitative analysis of types of bonds is possible. ‚ effects of solvent & molecular details complicate comparison

- Types of electron transitions: i) s, p, n electrons Single bonds usually too high excitation energy for most instruments (#185 nm) ‚ vacuum UV most compounds of atmosphere absorb in this range, so difficult to work with. ‚ usually concerned with functional groups with relatively low excitation energies (190 # l # 850 nm). - Types of electron transitions: i) s, p, n electrons Sigma (s) – single bond electron Low energy bonding orbital High energy anti-bonding orbital

Pi (p) – double bond electron Low energy bonding orbital High energy anti-bonding orbital Non-bonding electrons (n): don’t take part in any bonds, neutral energy level. Example: Formaldehyde

s  s* transition in vacuum UV ‚ n  s* saturated compounds with non-bonding electrons l ~ 150-250 nm e ~ 100-3000 ( not strong) ‚ n  p*, p  p* requires unsaturated functional groups (eq. double bonds) most commonly used, energy good range for UV/Vis l ~ 200 - 700 nm n  p* : e ~ 10-100 p  p*: e ~ 1000 – 10,000

Absorption Characteristics of Some Common Chromophores Example Solvent lmax (nm) emax Type of transition Alkene n-Heptane 177 13,000 pp* Alkyne 178 196 225 10,000 2,000 160 _ Carbonyl n-Hexane 186 280 180 293 1,000 16 Large 12 ns* np* Carboxyl Ethanol 204 41 Amido Water 214 60 Azo 339 5 Nitro CH3NO2 Isooctane 22 Nitroso C4H9NO Ethyl ether 300 665 100 20 Nitrate C2H5ONO2 Dioxane 270

Other Examples of Some Common Chromophores

< ethylenediamine < o-phenanthroline < NO2- < CN- Magnitude of D depends on: - charge on metal ion - position in periodic table - ligand field strength : I- < Br- < Cl- < F- < OH- < C2O42- ~ H2O < SCN- < NH3 < ethylenediamine < o-phenanthroline < NO2- < CN- D increases with increasing field strength, so wavelength decreases

C) Qualitative Analysis: Limited since few resolved peaks - unambiguous identification not usually possible. 2) Solvent can affect position and shape of curve. - polar solvents broaden out peaks, eliminates fine structure. Loss of fine structure for acetaldehyde when transfer to solvent from gas phase Also need to consider absorbance of solvent.

2) Solvent can affect position and shape of curve. - polar solvents broaden out peaks, eliminates fine structure. (a) Vapor Loss of fine structure for 1,2,4,5-tetrazine as solvent polarity increases (b) Hexane solution (c) Aqueous

3) Solvent can also absorb in UV-vis spectrum.

3) Can obtain some functional group information for certain types of compounds.. - weak band at 280-290 nm that is shifted to shorter l’s with an increase in polarity (solvent) implies a carbonyl group. acetone: in hexane, max = 279 nm ( = 15) in water, max = 264.5 nm - solvent effects due to stabilization or destabilization of ground or excited states, changing the energy gap. ‚ since most transitions result in an excited state that is more polar than the ground state - 260 nm with some fine structure implies an aromatic ring.

Benzene in heptane More complex ring systems shift to higher l’s (red shift) similar to conjugated alkenes

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

UV Spectroscopy Chromophores Organic Chromophores Alcohols, ethers, amines and sulfur compounds – in the cases of simple, aliphatic examples of these compounds the n  s* is the most often observed transition; like the alkane s  s* it is most often at shorter l than 200 nm Note how this transition occurs from the HOMO to the LUMO s*CN nN sp3 sCN

p* p 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

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

UV Spectroscopy Chromophores Organic Chromophores Carbonyls – n  p* transitions (~285 nm); p  p* (188 nm) p* It has been determined from spectral studies, that carbonyl oxygen more approximates sp rather than sp2 ! n p sCO transitions omitted for clarity

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

e 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 Hyperchromic e Hypsochromic Bathochromic Hypochromic 200 nm 700 nm

lmax nm e 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 35,000 465 125,000 n  p* 280 12 p  p* 189 900 n  p* 280 27 p  p* 213 7,100

Y2* f1 f2 Y1 p UV Spectroscopy Chromophores Substituent Effects 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, f1 and f2 from two sp2 hybrid carbons combine to form two MOs Y1 and Y2* in ethylene Y2* f1 f2 Y1 p

DE for the HOMO  LUMO transition is reduced UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes When we consider butadiene, we are now mixing 4 p orbitals giving 4 MOs of an energetically symmetrical distribution compared to ethylene Y4* Y2* Y3* Y2 Y1 p Y1 DE for the HOMO  LUMO transition is reduced

Energy UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes Extending this effect out to longer conjugated systems the energy gap becomes progressively smaller: Energy Lower energy = Longer wavelenghts ethylene butadiene hexatriene octatetraene

Energy UV Spectroscopy Chromophores Substituent Effects 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 p-system Y3* p* Y2 Energy p nA Y1

UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes Methyl groups also cause a bathochromic shift, even though they are devoid of p- or n-electrons This effect is thought to be through what is termed “hyperconjugation” or sigma bond resonance

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: Conjugated dienes Conjugated dienones Aromatic systems

s-trans s-cis UV Spectroscopy Structure Determination Dienes General Features For acyclic butadiene, two conformers are possible – s-cis and s-trans 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 s-trans s-cis

s-trans s-cis UV Spectroscopy Structure Determination Dienes General Features Two possible p  p* transitions can occur for butadiene Y2  Y3* and Y2  Y4* The Y2  Y4* transition is not typically observed: The energy of this transition places it outside the region typically observed – 175 nm For the more favorable s-trans conformation, this transition is forbidden The Y2  Y3* transition is observed as an intense absorption Y4* 175 nm –forb. 175 nm Y3* 217 nm 253 nm Y2 s-trans s-cis Y1

lmax = 217 253 220 227 227 256 263 nm UV Spectroscopy Structure Determination Dienes General Features The Y2  Y3* transition is observed as an intense absorption (e = 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: lmax = 217 253 220 227 227 256 263 nm

UV Spectroscopy Structure Determination Dienes 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 p  p* 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, Ultraviolet and Visible Spectroscopy, 3rd Ed., Butterworths, London, 1975)

Structure Determination Dienes Woodward-Fieser Rules - Dienes UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules - Dienes The rules begin with a base value for lmax 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 -OCOCH3 +0 -OR +6 -SR -Cl, -Br -NR2 +60

UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules - Dienes For example: Isoprene - acyclic butadiene = 217 nm one alkyl subs. + 5 nm 222 nm Experimental value 220 nm Allylidenecyclohexane - acyclic butadiene = 217 nm one exocyclic C=C + 5 nm 2 alkyl subs. +10 nm 232 nm Experimental value 237 nm

Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes There are two major types of cyclic dienes, with two different base values Heteroannular (transoid): Homoannular (cisoid): e = 5,000 – 15,000 e = 12,000-28,000 base lmax = 214 base lmax = 253 The increment table is the same as for acyclic butadienes with a couple additions: Group Increment Additional homoannular +39 Where both types of diene are present, the one with the longer l becomes the base

abietic acid levopimaric acid UV Spectroscopy Structure Determination Dienes 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: abietic acid levopimaric acid

UV Spectroscopy Structure Determination Dienes 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

UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes heteroannular diene = 214 nm 4 alkyl subs. (4 x 5) +20 nm 1 exo C=C + 5 nm 239 nm homoannular diene = 253 nm 4 alkyl subs. (4 x 5) +20 nm 1 exo C=C + 5 nm 278 nm

UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes Be careful with your assignments – three common errors: 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

UV Spectroscopy Structure Determination Enones General Features Carbonyls, as we have discussed have two primary electronic transitions: p* Remember, the p  p* transition is allowed and gives a high e, but lies outside the routine range of UV observation The n  p* transition is forbidden and gives a very low e, but can routinely be observed n p

UV Spectroscopy Structure Determination Enones General Features For auxochromic substitution on the carbonyl, pronounced hypsochromic shifts are observed for the n  p* transition (lmax): 293 nm 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 p  p* which extends conjugation and causes a bathochromic shift In most cases, this bathochromic shift is not enough to bring the p  p* transition into the observed range 279 235 214 204 204

UV Spectroscopy Structure Determination Enones General Features Conversely, if the C=O system is conjugated both the n  p* and p  p* bands are bathochromically shifted Here, several effects must be noted: the effect is more pronounced for p  p* if the conjugated chain is long enough, the much higher intensity p  p* band will overlap and drown out the n  p* band the shift of the n  p* transition is not as predictable For these reasons, empirical Woodward-Fieser rules for conjugated enones are for the higher intensity, allowed p  p* transition

UV Spectroscopy Structure Determination Enones General Features These effects are apparent from the MO diagram for a conjugated enone: Y4* p* p* Y3* n n Y2 p p Y1

Structure Determination Enones Woodward-Fieser Rules - Enones UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Group 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 a, b, g and higher 10, 12, 18 -OH 35, 30, 18 -OR a, b, g, d 35, 30, 17, 31 -O(C=O)R a, b, d 6 -Cl a, b 15, 12 -Br 25, 30 -NR2 b 95 Exocyclic double bond 5 Homocyclic diene component 39

Structure Determination Enones Woodward-Fieser Rules - Enones UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Aldehydes, esters and carboxylic acids have different base values than ketones Unsaturated system Base Value Aldehyde 208 With a or b alkyl groups 220 With a,b or b,b alkyl groups 230 With a,b,b alkyl groups 242 Acid or ester 217 Group value – exocyclic a,b double bond +5 Group value – endocyclic a,b bond in 5 or 7 membered ring

Structure Determination Enones Woodward-Fieser Rules - Enones UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Unlike conjugated alkenes, solvent does have an effect on lmax These effects are also described by the Woodward-Fieser rules Solvent correction Increment Water +8 Ethanol, methanol Chloroform -1 Dioxane -5 Ether -7 Hydrocarbon -11

UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Some examples – keep in mind these are more complex than dienes cyclic enone = 215 nm 2 x b- alkyl subs. (2 x 12) +24 nm 239 nm Experimental value 238 nm cyclic enone = 215 nm extended conj. +30 nm b-ring residue +12 nm d-ring residue +18 nm exocyclic double bond + 5 nm 280 nm Experimental 280 nm

UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Take home problem – can these two isomers be discerned by UV-spec allo-Eremophilone Eremophilone Problem Set 1: (text) – 1,2,3a,b,c,d,e,f,j, 4, 5, 6 (1st, 2nd and 5th pairs), 8a, b, c Problem Set 2: outside problems/key -Tuesday

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* p6* p4* p5* p2 p3 p1

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: E1u p6* B1u 200 nm (forbidden) p4* p5* B2u 180 nm (allowed) 260 nm (forbidden) p2 p3 A1g p1

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

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 --

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

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

Structure Determination Aromatic Compounds Substituent Effects UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects 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 Secondary Substituent lmax e -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 7,500 169 -C(O)OH 11,600 273 970 -C(O)O- 224 8,700 268 560

Structure Determination Aromatic Compounds Substituent Effects UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Substituents Capable of p-conjugation When the substituent is a p-chromophore, it can interact with the benzene p-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 Secondary Substituent lmax e -C(O)OH 230 11,600 273 970 -C(O)O- 224 8,700 268 560

UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Electron-donating and electron-withdrawing effects No matter what electronic influence a group exerts, the presence shifts the primary absorption band to longer l Electron-withdrawing groups exert no influence on the position of the secondary absorption band Electron-donating groups increase the l and e of the secondary absorption band

Structure Determination Aromatic Compounds Substituent Effects UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Electron-donating and electron-withdrawing effects Primary Secondary Substituent lmax e -H 203.5 7,400 254 204 -CH3 207 7,000 261 225 -Cl 210 264 190 -Br 7,900 192 -OH 211 6,200 270 1,450 -OCH3 217 6,400 269 1,480 -NH2 230 8,600 280 1,430 -CN 224 13,000 271 1,000 C(O)OH 11,600 273 970 -C(O)H 250 11,400 -C(O)CH3 9,800 -NO2 7,800 Electron donating Electron withdrawing

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:

UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects 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 lmax has been developed This is slightly less accurate than the Woodward-Fieser rules, but can usually predict within an error of 5 nm

Structure Determination Aromatic Compounds Substituent Effects UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Di-substituted and multiple group effects Parent Chromophore lmax R = alkyl or ring residue 246 R = H 250 R = OH or O-Alkyl 230 Substituent increment G o m p Alkyl or ring residue 3 10 -O-Alkyl, -OH, -O-Ring 7 25 -O- 11 20 78 -Cl -Br 2 15 -NH2 13 58 -NHC(O)CH3 45 -NHCH3 73 -N(CH3)2 85

UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects 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

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 400 500 600 700 800 l, nm Violet 400-420 Indigo 420-440 Blue 440-490 Green 490-570 Yellow 570-585 Orange 585-620 Red 620-780

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”

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

UV Spectroscopy Visible Spectroscopy Color General Likewise: lmax for lycopene is at 474 – in the near blue region of the spectrum – this is absorbed, the compliment is now red lmax for indigo is at 602 – in the orange region of the spectrum – this is absorbed, the compliment is now indigo!

UV Spectroscopy Visible Spectroscopy Color General One of the most common class of colored organic molecules are the azo dyes: 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!

UV Spectroscopy Visible Spectroscopy Color General These materials are some of the more familiar colors of our “environment”

The colors of M&M’s Bright Blue Royal Blue Orange-red Lemon-yellow Common Food Uses Beverages, dairy products, powders, jellies, confections, condiments, icing. Royal Blue Baked goods, cereals, snack foods, ice-cream, confections, cherries. Orange-red Gelatins, puddings, dairy products, confections, beverages, condiments. Lemon-yellow Custards, beverages, ice-cream, confections, preserves, cereals. Orange Cereals, baked goods, snack foods, ice-cream, beverages, dessert powders, confections

UV Spectroscopy Visible Spectroscopy Color 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.

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

Electronic Spectroscopy Chemistry 330 Chapter 17 Electronic Spectroscopy

Absorption and Emission The absorption spectrum of chlorophyll in the visible region. Absorbs in the red and blue regions, and that green light is not absorbed.

The Franck-Condon Principle The most intense vibronic transition is from the ground vibrational state to the vibrational state lying vertically above it. Transitions to other vibrational levels occur with lower intensity.

The Q-M Version A molecule will undergo a transition to the upper vibrational state when the upper state wavefunction most closely resembles the vibrational wavefunction of the vibrational ground state of the lower electronic state.

Types of Transitions Chromophore – a group with a characteristic optical absorption Various types of transition are posssible A d-d transition Vibronic transitions Charge transfer transitions  - * and n - * transitions

A d-d Transition Ligand field theory can be used to describe the electronic absorption spectrum of e.g. [Ti(OH2)6]3+ in aqueous solution. eg o t2g

A Typical Spectrum o  20 000 cm-1 for the complex [Ti(OH2)6]3+ in aqueous solution. Transitions usually occur in the visible region

Forbidden Transitions and Vibronic Spectra A d - d transition is parity-forbidden because it corresponds to a g - g transition. A vibration of the molecule can destroy the inversion symmetry The removal of the centre of symmetry gives rise to a vibronically allowed transition.

Transition Involving -electrons A C=C double bond acts as a chromophore. One of its important transitions is the *   transition The electron is promoted from a  MO orbital to the corresponding antibonding orbital.

Transition Involving -electrons (cont’d) A carbonyl (CO) group acts as a chromophore primarily on account of the excitation of a nonbonding O lone-pair electron to an antibonding CO  orbital.

Fluorescence vs. Phosphorescence The empirical distinction between fluorescence and phosphorescence Fluorescence is extinguished immediately when the the exciting source is removed, Phosphorescence continues with relatively slowly diminishing intensity.

Fluorescence The sequence of steps leading to fluorescence. The upper vibrational states undergo radiationless decay by giving up energy to the surroundings. Radiative transition then occurs from the vibrational ground state of the upper electronic state.

Absorption vs. Fluorescence An absorption spectrum (spectrum a) shows a vibrational structure characteristic of the upper state. A fluorescence spectrum (spectrum b) shows a structure characteristic of the lower state. 0,0 bands

Solvent Influences The solvent can shift the fluorescence spectrum relative to the absorption spectrum. Before fluorescence occurs, the solvent molecules relax into a new arrangement, and that arrangement is preserved during the subsequent radiative transition.

Phosphorescence The sequence of steps leading to phosphorescence Intersystem crossing - the switch from a singlet state to a triplet state brought about by spin-orbit coupling. The triplet state –ground state transition is spin-forbidden.

A Jablonski Diagram for Naphthalene Displays of the relative positions of the electronic energy levels of a molecule. IC - internal conversion ISC - intersystem crossing.) Vibrational levels of states of a given electronic state lie above each other, but the relative horizontal locations of the columns bear no relation to the nuclear separations in the states. The ground vibrational states of each electronic state are correctly located vertically but the other vibrational states are shown only schematically.

Continuum Absorption When absorption occurs to unbound states of the upper electronic state, the molecule dissociates and the absorption is a continuum. Below the dissociation limit - normal vibrational structure.

Dissociative States When a dissociative state crosses a bound state, molecules excited to levels near the crossing may dissociate. This process is called predissociation, and is detected in the spectrum as a loss of vibrational structure that resumes at higher frequencies.

A Three Level Laser The transitions involved in one kind of three-level laser. The pumping pulse populates the intermediate state I, which in turn populates the laser state A. Laser transition is the stimulated emission A  X.

A Four–Level Laser The transitions involved in a four-level laser. Because the laser transition terminates in an excited state (A), the population inversion betweeen A and A' is much easier to achieve.

The Steps Leading to Laser Action The Boltzmann population of states, with more atoms in the ground state. When the initial state absorbs, the populations are inverted (the atoms are pumped to the excited state).

Laser Action A cascade of radiation then occurs, One emitted photon stimulates another atom to emit The radiation is coherent (phases in step).

Q-Switching The principle of Q-switching. The excited state is populated while the cavity is nonresonant. The resonance characteristics are suddely restored, and the stimulated emission emerges in a giant pulse.

A Mode-Locked Laser The output of a mode-locked laser consists of a stream of very narrow pulses separated by an interval equal to the time it takes for light to make a round trip inside the cavity.

The Requirements for Laser Action A summary of the features needed for efficient laser action.

The Principles of Photoelectron Spectroscopy An incoming photon carries an energy h; an energy Ii is needed to remove an electron from an orbital i, and the difference appears as the kinetic energy of the electron.

The Photoelectron Spectrometer A photoelectron spectrometer consists of a source of ionizing radiation a helium discharge lamp for UPS X-ray source for XPS Electrostatic analyser Electron detector The deflection of the electron path caused by the analyser depends on the speed at which they are ejected from the sample.

The Photoelectron Spectrum of HBr The lowest ionization energy bands () correspond to the ionization of a Br lone-pair electron. The higher ionization energy band () corresponds to the ionization of a bonding electron. The structure on the latter is due to the vibrational excitation of HBr+ that results from the ionization.

Fall 2005 Chapter 7: UV Spectroscopy UV & electronic transitions Usable ranges & observations Selection rules Band Structure Instrumentation & Spectra Beer-Lambert Law Application of UV-spec CHMBD 449 – Organic Spectral Analysis

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 g-rays X-rays UV IR Microwave Radio Visible

UV Spectroscopy Introduction The Spectroscopic Process 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

UV Spectroscopy Introduction 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)

Observed electronic transitions Here is a graphical representation UV Spectroscopy Introduction Observed electronic transitions Here is a graphical representation s* Unoccupied levels p* Atomic orbital Atomic orbital Energy n Occupied levels p s Molecular orbitals

UV Spectroscopy Introduction Observed electronic transitions From the molecular orbital diagram, there are several possible electronic transitions that can occur, each of a different relative energy: s* s p n s* p* alkanes carbonyls unsaturated cmpds. O, N, S, halogens p* Energy n p s

UV Spectroscopy Introduction 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 p n s* p* alkanes carbonyls unsaturated cmpds. O, N, S, halogens 150 nm 170 nm 180 nm √ - if conjugated! 190 nm 300 nm √

UV Spectroscopy Introduction Selection Rules Not all transitions that are possible are observed 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

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

UV Spectroscopy Introduction Band Structure When these energy levels are superimposed, the effect can be readily explained – any transition has the possibility of being observed E1 Energy E0

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: log(I0/I) = A I0 I UV-VIS sources sample 200 700 l, nm detector monochromator/ beam splitter optics I0 I0 reference

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

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 Diode array UV-VIS sources sample Polychromator – entrance slit and dispersion device

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):

UV Spectroscopy Instrumentation and Spectra Instrumentation – Sample Handling 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 Since spectra are only obtained up to 200 nm, solvents typically only need to lack conjugated p systems or carbonyls Common solvents and cutoffs: acetonitrile 190 chloroform 240 cyclohexane 195 1,4-dioxane 215 95% ethanol 205 n-hexane 201 methanol 205 isooctane 195 water 190

UV Spectroscopy Instrumentation and Spectra Instrumentation – Sample Handling Additionally solvents must preserve the fine structure (where it is actually observed in UV!) where possible 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)

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

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…

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

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

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

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

UV Spectroscopy Chromophores Organic Chromophores Alcohols, ethers, amines and sulfur compounds – in the cases of simple, aliphatic examples of these compounds the n  s* is the most often observed transition; like the alkane s  s* it is most often at shorter l than 200 nm Note how this transition occurs from the HOMO to the LUMO s*CN nN sp3 sCN

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

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

UV Spectroscopy Chromophores Organic Chromophores Carbonyls – n  p* transitions (~285 nm); p  p* (188 nm) p* It has been determined from spectral studies, that carbonyl oxygen more approximates sp rather than sp2 ! n p sCO transitions omitted for clarity

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

General – Substituents may have any of four effects on a chromophore 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 Hyperchromic e Hypsochromic Bathochromic Hypochromic 200 nm 700 nm

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 35,000 465 125,000 n  p* 280 12 p  p* 189 900 n  p* 280 27 p  p* 213 7,100

UV Spectroscopy Chromophores Substituent Effects 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, f1 and f2 from two sp2 hybrid carbons combine to form two MOs Y1 and Y2* in ethylene Y2* f1 f2 Y1 p

UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes When we consider butadiene, we are now mixing 4 p orbitals giving 4 MOs of an energetically symmetrical distribution compared to ethylene Y4* Y2* Y3* Y2 Y1 p Y1 DE for the HOMO  LUMO transition is reduced

UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes Extending this effect out to longer conjugated systems the energy gap becomes progressively smaller: Energy Lower energy = Longer wavelengths ethylene butadiene hexatriene octatetraene

UV Spectroscopy Chromophores Substituent Effects 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 p-system Y3* p* Y2 Energy p nA Y1

UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes Methyl groups also cause a bathochromic shift, even though they are devoid of p- or n-electrons This effect is thought to be through what is termed “hyperconjugation” or sigma bond resonance

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: Conjugated dienes Conjugated dienones Aromatic systems

UV Spectroscopy Structure Determination Dienes General Features For acyclic butadiene, two conformers are possible – s-cis and s-trans 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 s-trans s-cis

UV Spectroscopy Structure Determination Dienes General Features Two possible p  p* transitions can occur for butadiene Y2  Y3* and Y2  Y4* The Y2  Y4* transition is not typically observed: The energy of this transition places it outside the region typically observed – 175 nm For the more favorable s-trans conformation, this transition is forbidden The Y2  Y3* transition is observed as an intense absorption Y4* 175 nm –forb. 175 nm Y3* 217 nm 253 nm Y2 s-trans s-cis Y1

UV Spectroscopy Structure Determination Dienes General Features The Y2  Y3* transition is observed as an intense absorption (e = 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: lmax = 217 253 220 227 227 256 263 nm

UV Spectroscopy Structure Determination Dienes 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 p  p* 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, Ultraviolet and Visible Spectroscopy, 3rd Ed., Butterworths, London, 1975)

Structure Determination Dienes Woodward-Fieser Rules - Dienes UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules - Dienes The rules begin with a base value for lmax 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 -OCOCH3 +0 -OR +6 -SR -Cl, -Br -NR2 +60

UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules - Dienes For example: Isoprene - acyclic butadiene = 217 nm one alkyl subs. + 5 nm 222 nm Experimental value 220 nm Allylidenecyclohexane - acyclic butadiene = 217 nm one exocyclic C=C + 5 nm 2 alkyl subs. +10 nm 232 nm Experimental value 237 nm

Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes There are two major types of cyclic dienes, with two different base values Heteroannular (transoid): Homoannular (cisoid): e = 5,000 – 15,000 e = 12,000-28,000 base lmax = 214 base lmax = 253 The increment table is the same as for acyclic butadienes with a couple additions: Group Increment Additional homoannular +39 Where both types of diene are present, the one with the longer l becomes the base

UV Spectroscopy Structure Determination Dienes 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: abietic acid levopimaric acid

UV Spectroscopy Structure Determination Dienes 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

UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes heteroannular diene = 214 nm 4 alkyl subs. (4 x 5) +20 nm 1 exo C=C + 5 nm 239 nm homoannular diene = 253 nm 4 alkyl subs. (4 x 5) +20 nm 1 exo C=C + 5 nm 278 nm

UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes Be careful with your assignments – three common errors: 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

UV Spectroscopy Structure Determination Enones General Features Carbonyls, as we have discussed have two primary electronic transitions: p* Remember, the p  p* transition is allowed and gives a high e, but lies outside the routine range of UV observation The n  p* transition is forbidden and gives a very low e, but can routinely be observed n p

UV Spectroscopy Structure Determination Enones General Features For auxochromic substitution on the carbonyl, pronounced hypsochromic shifts are observed for the n  p* transition (lmax): 293 nm 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 p  p* which extends conjugation and causes a bathochromic shift In most cases, this bathochromic shift is not enough to bring the p  p* transition into the observed range 279 235 214 204 204

UV Spectroscopy Structure Determination Enones General Features Conversely, if the C=O system is conjugated both the n  p* and p  p* bands are bathochromically shifted Here, several effects must be noted: the effect is more pronounced for p  p* if the conjugated chain is long enough, the much higher intensity p  p* band will overlap and drown out the n  p* band the shift of the n  p* transition is not as predictable For these reasons, empirical Woodward-Fieser rules for conjugated enones are for the higher intensity, allowed p  p* transition

UV Spectroscopy Structure Determination Enones General Features These effects are apparent from the MO diagram for a conjugated enone: Y4* p* p* Y3* n n Y2 p p Y1

Structure Determination Enones Woodward-Fieser Rules - Enones UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Group 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 a, b, g and higher 10, 12, 18 -OH 35, 30, 18 -OR a, b, g, d 35, 30, 17, 31 -O(C=O)R a, b, d 6 -Cl a, b 15, 12 -Br 25, 30 -NR2 b 95 Exocyclic double bond 5 Homocyclic diene component 39

Structure Determination Enones Woodward-Fieser Rules - Enones UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Aldehydes, esters and carboxylic acids have different base values than ketones Unsaturated system Base Value Aldehyde 208 With a or b alkyl groups 220 With a,b or b,b alkyl groups 230 With a,b,b alkyl groups 242 Acid or ester 217 Group value – exocyclic a,b double bond +5 Group value – endocyclic a,b bond in 5 or 7 membered ring

Structure Determination Enones Woodward-Fieser Rules - Enones UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Unlike conjugated alkenes, solvent does have an effect on lmax These effects are also described by the Woodward-Fieser rules Solvent correction Increment Water +8 Ethanol, methanol Chloroform -1 Dioxane -5 Ether -7 Hydrocarbon -11

UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Some examples – keep in mind these are more complex than dienes cyclic enone = 215 nm 2 x b- alkyl subs. (2 x 12) +24 nm 239 nm Experimental value 238 nm cyclic enone = 215 nm extended conj. +30 nm b-ring residue +12 nm d-ring residue +18 nm exocyclic double bond + 5 nm 280 nm Experimental 280 nm

UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Take home problem – can these two isomers be discerned by UV-spec allo-Eremophilone Eremophilone Problem Set 1: (text) – 1,2,3a,b,c,d,e,f,j, 4, 5, 6 (1st, 2nd and 5th pairs), 8a, b, c Problem Set 2: outside problems/key -Tuesday

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* p6* p4* p5* p2 p3 p1

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: E1u p6* B1u 200 nm (forbidden) p4* p5* B2u 180 nm (allowed) 260 nm (forbidden) p2 p3 A1g p1

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

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 --

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

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

Structure Determination Aromatic Compounds Substituent Effects UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects 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 Secondary Substituent lmax e -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 7,500 169 -C(O)OH 11,600 273 970 -C(O)O- 224 8,700 268 560

Structure Determination Aromatic Compounds Substituent Effects UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Substituents Capable of p-conjugation When the substituent is a p-chromophore, it can interact with the benzene p-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 Secondary Substituent lmax e -C(O)OH 230 11,600 273 970 -C(O)O- 224 8,700 268 560

UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Electron-donating and electron-withdrawing effects No matter what electronic influence a group exerts, the presence shifts the primary absorption band to longer l Electron-withdrawing groups exert no influence on the position of the secondary absorption band Electron-donating groups increase the l and e of the secondary absorption band

Structure Determination Aromatic Compounds Substituent Effects UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Electron-donating and electron-withdrawing effects Primary Secondary Substituent lmax e -H 203.5 7,400 254 204 -CH3 207 7,000 261 225 -Cl 210 264 190 -Br 7,900 192 -OH 211 6,200 270 1,450 -OCH3 217 6,400 269 1,480 -NH2 230 8,600 280 1,430 -CN 224 13,000 271 1,000 C(O)OH 11,600 273 970 -C(O)H 250 11,400 -C(O)CH3 9,800 -NO2 7,800 Electron donating Electron withdrawing

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:

UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects 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 lmax has been developed This is slightly less accurate than the Woodward-Fieser rules, but can usually predict within an error of 5 nm

Structure Determination Aromatic Compounds Substituent Effects UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Di-substituted and multiple group effects Parent Chromophore lmax R = alkyl or ring residue 246 R = H 250 R = OH or O-Alkyl 230 Substituent increment G o m p Alkyl or ring residue 3 10 -O-Alkyl, -OH, -O-Ring 7 25 -O- 11 20 78 -Cl -Br 2 15 -NH2 13 58 -NHC(O)CH3 45 -NHCH3 73 -N(CH3)2 85

UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects 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

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 400 500 600 700 800 l, nm Violet 400-420 Indigo 420-440 Blue 440-490 Green 490-570 Yellow 570-585 Orange 585-620 Red 620-780

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”

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

UV Spectroscopy Visible Spectroscopy Color General Likewise: lmax for lycopene is at 474 – in the near blue region of the spectrum – this is absorbed, the compliment is now red lmax for indigo is at 602 – in the orange region of the spectrum – this is absorbed, the compliment is now indigo!

UV Spectroscopy Visible Spectroscopy Color General One of the most common class of colored organic molecules are the azo dyes: 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!

UV Spectroscopy Visible Spectroscopy Color General These materials are some of the more familiar colors of our “environment”

The colors of M&M’s Bright Blue Royal Blue Orange-red Lemon-yellow Common Food Uses Beverages, dairy products, powders, jellies, confections, condiments, icing. Royal Blue Baked goods, cereals, snack foods, ice-cream, confections, cherries. Orange-red Gelatins, puddings, dairy products, confections, beverages, condiments. Lemon-yellow Custards, beverages, ice-cream, confections, preserves, cereals. Orange Cereals, baked goods, snack foods, ice-cream, beverages, dessert powders, confections

UV Spectroscopy Visible Spectroscopy Color 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.

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

UV-visible molecular absorption spectroscopy Chemistry 243

Transmission and absorbance and losses The reduction in the intensity of light transmitted through a sample can be used to quantitate the amount of an unknown material.

Beer’s Law Really: Al = elbc Quantitative relationship between absorbance and concentration of analyte See derivation in text (Skoog: pages 337-338) Absorption is additive for mixtures Really: Al = elbc Beer’s Law is always wavelength-specific

Limitations and deviations from Beer’s Law Real limitations Non-linearities due to intermolecular interactions Self aggregation effects and electrolyte effects Apparent Dynamic dissociation or association of analyte Instrumental Polychromatic radiation Different molar absorptivities at different wavelength leads to non-linearities in Beer’s Law Stray radiation Mistmatched cells Non-zero intercept in calibration curve How might one avoid?

How to make a UV-vis absorption measurement Make a 0%T (dark current) measurement Make a 100%T (blank) measurement Measure %T of sample Determine %T ratio and thus the absorbance value

Instrumental noise Precision of measurement is limited by instrumental noise sources

Use proper slit widths Resolution improves with narrower slit width, but power decreases as square of slit width. 10-fold narrower slit gives 100x less radiant power General rule: Use the widest slit that gives required resolution.

Light sources for UV-vis Deuterium lamp Most common UV source Arc between oxide-coated filament and metal electrode Low voltage and low pressure of D2 Aperture gives 1-1.5 mm spot Continuum from 190-400 nm, emission lines >400nm

Light sources for UV-vis, continued Tungsten filament Most common visible and NIR source Blackbody radiator useful from 350-2500 nm Power varies as (operating voltage)4; need stable power supply! Tungsten-halogen sources can operate at higher temperatures and give off more UV light.

Light sources for UV-vis, continued2 LEDs 375-1000 nm Semi-monochromatic (20-50 nm FWHM) “White” LEDs use phosphor to give 400-800 nm continuum Keychain flashlights Xenon arc lamps Very intense source Continuum from 200-1000 nm, peaking at 500 nm

Instrument configurations Single-beam Double-beam Multichannel

Single-beam UV-vis spectrometers Skoog, Fig. 13-13 Good light throughput, but what if the source power fluctuates?

Double-beam in time UV vis spectrometers Beam is split in two, but measured by same detector “in time” because the beam appears in 2 places over one cycle in time - Sample - Reference Sample Reference What if the source power fluctuates? Skoog, Fig. 13-13

Double-beam in space UV-vis spectrometers Beam is split into two paths and measured by matched detectors Difficult to find perfectly matched detectors “in space” because two beams are always present in space Continuous Reference What if the source power fluctuates? Continuous Sample

Cary 100 double beam spectrometer - Sample - Dark - Reference

Cary 300 double-dispersing spectrophotometer Why does double dispersion help with extending absorption to ~5.0 absorbance units? Two gratings Reduced stray light 0.00008% or less Improved spectral resolution Bandwidth < 4 nm If Abs = 5.0, %T = ?

Multichannel UV-vis spectrometers Dispersing optic (grating or prism) used to separate different wavelengths in space. Detection with diode array or CCD Fast acquisition of entire spectrum

Diode array spectrophotometers Fairly inexpensive, but good quality fiber optic models available for ~$3000. Ocean Optics StellarNet

Diode array spectrophotometers http://www.oceanoptics.com/products/usb4000.asp 89 mm 3.5 inches 250 specta per sec

Reflective dip probes

What is UV-visible absorption measuring? The absorption of a photon generates an electronic excited state UV-vis energy often matches up with transitions of bonding electrons Often relatively short lifetimes (1-10 nsec) Relaxation can occur non-radiatively or by emission of radiation (fluorescence or phosphorescence)

Absorption signatures of various organic functional groups Commonly observed transitions are np* or pp* Chromophores have unsaturated functional groups Rotational and vibrational transitions add detail to spectra Single bond excitation energies (ns*) are in vacuum UV (l < 185 nm) and have very low molar absorptivities e normalized with respect to path length and concentration

Absorption signatures of various organic functional groups, continued Conjugation causes shift to longer wavelength pp* transitions more 10-100x or more intense than np* Nonbonding electrons of heteroatoms in saturated compounds can give UV absorbance signature. Note distinct lmax values

Spectra of inorganic (metal and non-metal) ions and ionic complexes Inorganic anions have broad UV absorption bands from non-bonding electrons. Transition metal ions and complexes absorb visible light upon excitation between filled and unfilled d-orbitals. Dependent upon oxidation state and coordination environment.

Spectra of lanthanide and actinide ions Lanthanide and actinide ions absorptions come from excitation of 4f and 5f electrons. f electrons are shielded from s, p, and d orbitals and have narrow absorption bands

Charge-transfer complexes Electron donor absorbs light and transfers to acceptor. Internal red-ox process Typically very large molar absorptivities (e>10,000) Metal-to-ligand charge transfers (MLCT) Ligand-to-metal charge transfer (LMCT) http://www.piercenet.com/browse.cfm?fldID=876562B0-5056-8A76-4E0C-B764EAB3A339

Environmental effects The environment that the analyte is in can have profound effect on the observed spectrum In the gas phase, rotational and vibrational fine structure can be observed given adequate spectral bandwidth. In solid form or in solution, molecules cannot rotate as freely and differences in rotational energy level are not observable. Solvent molecules can also lead to a loss of vibrational detail in the absorbance spectrum. The visible absorption spectrum of sym-tetrazine: I, at room temperature in the vapour; II, at 77o K in a 5 : 1 isopentane-methylcyclohexane glass, III, in cyclohexane; and IV, in aqueous solution at room temperature. J. Chem. Soc., 1959, 1263-1268.

Solvatochromism The polarity of solvents can preferentially stabilize the ground or excited state leading to different energy level gaps and thus a solvent-dependent absorption spectrum. acetone isopropanol ethanol http://scienceblogs.com/moleculeoftheday/2007/02/reichardts_dye_solvatochromic.php http://www.uni-regensburg.de/Fakultaeten/nat_Fak_IV/Organische_Chemie/Didaktik/Keusch/p28_neg_sol-e.htm

Solvatochromism, continued Positive solvatochromism (red shift) Bathochromic Negative solvatochromism (blue shift) Hypsochromic Resonance structures of 4,4'-bis(dimethylamino)fuchsone http://www.chemie.uni-regensburg.de/Organische_Chemie/Didaktik/Keusch/D-pos_sol-e.htm http://www.uni-regensburg.de/Fakultaeten/nat_Fak_IV/Organische_Chemie/Didaktik/Keusch/p28_neg_sol-e.htm

Qualitative versus quantitative analysis via UV-vis absorption What are the objectives of qualitative versus quantitative UV-visible absorption spectroscopy? How might the application guide slit width selection? Large slit width = good sensitivity but poor resolution Small slit width = poor sensitivity but good resolution Qualitative work needs __?? Quantitative work needs __?? Visible region absorbance spectrum for cytochrome c with spectral bandwidths of (1) 20 nm, (2) 10 nm, (3) 5 nm, and (4) 1 nm.

Attributes of UV-visible absorption for quantitative analysis Applicable to organic and inorganic species Good detection limits: 10-100 mM or better Possible need for larger slit widths to achieve best sensitivities Moderate to high selectivity Accuracy: 1-3% or better Ease and convenience ($$$) of data acquisition

Considerations for using UV-vis for quantitative measurements Directly monitor absorbing analytes; usually non-destructive Can use reagents that react with colorless analyte to generate measureable species Greatly increase molar absorptivity Thiocyanate (Fe, Co, Mo), H2O2 (Ti, V, Cr), iodide (Bi, Pd, Te) Monitor at wavelength of max absorption, max at lmax Greatest change in absorbance per unit concentration Absorbance least sensitive to a small change in wavelength Relaxes requirement on instrument to stringently achieve the exact same wavelength UV-visible absorbance sensitive to environment, pH, temperature, high electrolyte concentration, interfering species. Be careful with standards Use matched cells.

Calibration and mixture analysis Generate calibration curve (linear) using external standards Must use multiple standards Standards hopefully match sample matrix Matrix matching is hard—consider using standard addition. Mixtures are additive Need to monitor at as many wavelengths as components to be analyzed. Requirement of solving multiple equations with multiple unknowns.

Color Analysis with Visible Spectra The visible region of a UV-Visible spectrum can be decomposed into a color analysis (typically three numbers) by simple calculations Involves multiplying the visible portion of the spectrum by color functions and then taking the total area of the spectrum as a single number Tristimulus values, which mimic the eye, are generally used and then other values are determined from these algebraically http://www.zeiss.de/c12567bb00549f37/Contents-Frame/80bd2fe43b50aa3ec125782c00597389

Diffuse Reflectance UV-Visible Spectroscopy of Solids Solid powders can be studied using a diffuse reflectance (DR) accessory either neat or diluted in a non-absorbing powder

Diffuse Reflectance UV-Visible Spectroscopy of Solids Typical diffuse reflectance spectrum of cyanocobalamin (vitamin B12), diluted to 5% w/w in MgO

Prediction of UV-Visible Spectra with Quantum Calculations: Time-dependent DFT TDDFT: Time-dependent density functional theory currently provides accurate predictions of UV-visible spectra for organic molecules J. Mol. Struct. 2010, 984, 246–261, ttp://dx.doi.org/10.1016/j.molstruc.2010.09.036

Plane (or Linearly) Polarized Light If the electric vector of an EM wave points in the same direction as that of the wave propagating through a medium, the light is said to be linearly polarized Birefringence – different indices of refraction for difference polarizations. Dichroism – the preferential absorption of some polarization. (Example – a crystal absorbing x-polarized radiation and not y-polarized radiation). Polarization can change on reflection from a dielectric surface (a mirror). Two mirrors can be used to change polarized light from the x-axis to the y-axis, see pg 150 of “Building scientific apparatus” Figure from Sears, et al., “University Physics”, 7th Ed., 1988

Polarimetry and Optical Rotation A polarimeter measures the angle of rotation of linearly polarized monochromatic light as it passes through a sample Source: sodium arc lamp (589 nm), now commonly replaced with a yellow LED Two polarizers before and after the sample. One is fixed and the other is rotated to find the maximum light transmitted, and the rotation is recorded. Result is a single number, e.g. -10.02, the specific rotation What happens when we vary the wavelength?

Optical Rotation and ORD The rotation of plane polarized light by molecules: Eliel et al., “Stereochemistry of Organic Compounds”, p. 997. R. P Feynman, et al., “The Feynman Lectures on Physics”, 1963, Addison-Wesley. p. 33-6

Optical Rotatory Dispersion (ORD) The measurement of specific rotation as a function of wavelength, in the absence of absorption, is monotonic (and governed by the Fresnel equation) In the vicinity of an absorption, one obtains “anomalous dispersion”

UV-Visible Circular Dichroism UV-visible or electronic circular dichroism (ECD or just CD) is the study of differential absorption of polarized UV-Visible radiation by chiral molecules. CD measures the difference between LCPL and RCPL Beer’s law for CD: A = bc Where  = (LPCL - RPCL)  is the molar absorptivity (cm-1 M-1) A is absorption For more information see pg. 127 of Skoog, et al. LHCP light can be converted to RHCP by reflection. See Eliel, et al. Stereochemistry of Organic Compounds, pg. 1003.

Circularly-Polarized UV-Visible Radiation Circularly-polarized UV-visible radiation is made by mixing two orthogonal electric field components 90 degrees out of phase. In practice, a quartz crystal is subjected to mechanical stress and (via the piezoelectric effect) causes circular polarization of the light For more information see pg. 127 of Skoog, et al. LHCP light can be converted to RHCP by reflection. Animation from http://www.bip.bham.ac.uk/osmart/bcm201_cd/cd_movie/index.html

UV-Visible Circular Dichroism A typical UV-Visible CD spectrometer, the Jasco J-715

Electronic Circular Dichroism CD spectra of (1S)-(+)-10-camphorsulfonic acid and (1R)-(+)-10-camphorsulfonic acid (ammonium salts) in H2O

TDDFT Calculations TDDFT calculations have largely replaced empirical rules. Example: (1R)-(+)-10-camphorsulfonic acid (ammonium salts) and its isomer calculated without solvation:

Electronic Circular Dichroism Variable temperatuer CD spectra of an orally-bioavailable PTH mimetic peptide, showing conformational changes: 1 H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu- 16 Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp- 31 Val-(NH2) Anal. Chem. 2012, 84, 4357-4372, http://dx.doi.org/10.1021/ac203478r

Electronic Circular Dichroism ECD has extensive applications to structural analysis in proteins, antibodies, and other biopolymers N. Sreerama and R. W. Woody, Meth. Enzymology, 2004, 383, 318-351.

Electronic Circular Dichroism Different protein conformations give rise to different spectra CD spectra are numerically fitted to extract conformational population N. Sreerama and R. W. Woody, Meth. Enzymology, 2004, 383, 318-351.

Hyphenated Circular Dichroism Experiments Example: Related atropoisomeric compounds studied in stopped-flow LC-CD experiments T. J. Edkins and D. R. Bobbitt, Anal. Chem., 2001, 73, 488A-496A G. Bringmann, et al., Anal. Chem., 1999, 71, 2678-2686.

The Cotton Effect The Cotton effect: An extrema in the ECD spectrum Or, a zero-crossing in the ORD spectrum

Other Notes on Electronic Circular Dichroism Background signals – UV absorbance that does not depend on the polarization constitutes the background (Dynamic Reserve). DR = A/A = / = /(LPCL - RPCL)  is the molar absorptivity (cm-1 M-1) A is absorption DR values of 2 x104 are possible Electronic background suppression is almost always used instead of optical background suppression (technical design issues)

Elliptically Polarized Light Combining left and right circularly polarized waves of unequal amplitudes = elliptically polarized light Basis of ellipsometry – a surface analysis method used to study: Layer/film thickness Optical constants (refractive index and extinction coefficient) Surface roughness Composition Optical anisotropy

Further Reading Optional: J. Cazes, Ed. Ewing’s Analytical Instrumentation Handbook, 3rd Edition, 2005, Marcel Dekker, Chapters 5 and 6. D. A. Skoog, F. J. Holler and S. R. Crouch, Principles of Instrumental Analysis, 6th Edition, 2006, Brooks-Cole, Chapters 13 and 14. D. H. Williams and I. Fleming, “Spectroscopic Methods in Organic Chemistry”, McGraw-Hill (1966). D. A. Lightner and J. E. Gurst, “Organic Conformational Analysis and Stereochemistry from Circular Dichroism Spectroscopy,” Wiley-VCH, 2000.

Molecular UV-Vis Spectroscopy: Quantum Theory UV-Visible spectra and the states involved in electronic transitions can be calculated with theories ranging from Huckel to ab initio/DFT. Example:   * transitions responsible for ethylene UV absorption at ~170 nm calculated with ZINDO semi-empirical excited-states methods (Gaussian 03W): HOMO u bonding molecular orbital LUMO g antibonding molecular orbital Huckel and extended Huckel.

Instrument noise: noise - short term baseline fluctuations, which decrease the precision of the analysis -can not measure A precisely - the various sources of noise each cause some uncertainty in the absorbance measurement and can be treated as individual standard deviations. 1) 0%T noise: - noise when light beam is blocked - seldom important - typically " 0.01%T 2) Readout Precision: - especially with a meter - typically " 0.5%T  1-3% error in concentration 3) Shot Noise: - occurs when e- transfers across a junction (like the space between cathode & anode in PMT). - causes random fluctuations in current since individual e- arrive at random times - increases with increase current (%T). Especially bad above 95%T.

Likely to be Important in 4) Flicker Noise: - noise from the lamp due to intensity changes important at high transmittances. 5) Cell positioning uncertainty: - not really noise, but affects precision - minor imperfections, scratches or dirt change %T - may be the major cause of imprecision Category Characterized by Typical Sources Likely to be Important in Case I ST = k1 Limited Readout resolution Inexpensive photometers and spectrophotometers having small meter scales Heat detector Johnson noise IR and near-IR spectrophotometers and photometers Dark current and amplifier noise Regions where source intensity and detector are low Case II ST = k2 rT2 +T Photon detector shot noise High-quality UV-visible spectrophotometers Case III ST = k3T Cell positioning uncertainties High-quality UV-visible and IR spectrophotometers Source flicker Inexpensive photometers and spectrophotometers

- at low %T, 0%T, noise and readout precision are important Taken together, these noise sources indicate that the intermediate absorbance and transmittance ranges should be used. - at low %T, 0%T, noise and readout precision are important - at high %T, shot and flicker noise are large. Keep A in range of 0.1 – 1.5 absorbance units (80 -3%T)

Quantum: the energy of a photon E = h  = c  h•c  E   E       (cm) short high Wavelength () Frequency () Energy (E) long low

Ultraviolet-Visible (UV-Vis) Spectroscopy  200 400 800 nm Recall bonding of a -bond

If blank absorbance too high: - dilute the sample But, If the blank absorbance is high, Po will decrease too much, the response will be slow and the results inaccurate l scan of substance Large blank absorbance If blank absorbance too high: - dilute the sample - use a different l where the analyte absorbs more relative to the interference. - use a different method of separation

b) Instrumental Deviations from Beer’s Law: - stray light (already discussed). - polychromatic light (more then a single l) ‚ since all instruments have a finite bandpass, a range of l’s are sent through the sample. ‚ e may be different for each l Deviation’s from Beer’s law at high concentration

Illustration of Deviation from Beer’s law: Let us say that exactly 2 wavelengths of light were entering the sample l = 254 nm e254 =10,000 l = 255 nm e255 = 5,000 let Po = 1 at both l’s What happens to the Beer’s law plot as c increases? A = A254 + A255 A = log Po/P (total) = log (Po254 + Po255)/(P254 + P255) At the individual l’s: A254 = e254bc = log Po254/P254 10ebc = Po254/P254 P254 = Po/10ebc = Po10-ebc For both together: A = log (Po254 + Po255)/(Po25410-e254bc + Po25510-e255bc)

Since Po254 = Po255 =1: A = 2.0/(Po25410-e254bc + Po25510-e255bc) A = 2.0/(10-10,000x1.0xc + 10-5000x1.0xc) C A (actual) A(expected) 10-6M 0.0075 10-5M 0.074 0.075 10-4M 0.068 0.75 10-3M 5.3 7.5 The results are the same for more l’s of light. The situation is worse for greater differences in e’s (side of absorption peak, broad bandpass) Always need to do calibration curve! Can not assume linearity outside the range of linearity curve!

b) Chemical Deviations from Beer’s Law: - Molar absorptivity change in solutions more concentrated than 0.01M ‚ due to molecular interactions ‚ Beer’s law assumes species are independent ‚ electrolytes may also cause this problem ‚ e is also affected by the index of refraction - association, dissociation, precipitation or reaction of analyte ‚ c in Beer’s law is the concentration of the absorbing species. ‚ commonly use the analytical concentration – concentration of all forms of the species. Ka phenolphthalein: HIn H+ + In- Red, l =600nm colorless If solution is buffered, then pH is constant and [HIn] is related to absorbance.

But, if unbuffered solution, equilibrium will shift depending on total analyte concentration example: if Ka = 10-4 HIn Ka H+ + In- CHIn [HIn] [In-] [HIn]/[In-] 10-5 8.5x10-7 9.2x10-6 0.0924 10-4 3.8x10-5 6.2x10-5 0.613 10-3 7.3x10-4 2.7x10-4 2.70 “Apparent” deviation since can be accounted for by chemical equilibrium

But, if unbuffered solution, equilibrium will shift depending on total analyte concentration example: if Ka = 10-4 HIn Ka H+ + In- Isosbestic point At the isosbestic point in spectra: A = eb([HIn] + [In-])

- worse for round cuvettes - use parallel cuvettes to help c) Non-constant b: - worse for round cuvettes - use parallel cuvettes to help B2 P2 P0 B1 P1 A = log10 Po/P = ebc

ii) d/f electrons (transition metal ions) ‚ Lanthanide and actinide series - electronic transition of 4f & 5f electrons - generally sharp, well-defined bands not affected by associated ligands ‚ 1st and 2nd transition metal series - electronic transition of 3d & 4d electrons - broad peaks Crystal-Field Theory In absence of external field d-orbitals are identical Energies of d-orbitals in solution are not identical Absorption involves e- transition between d- orbitals In complex, all orbitals increase in energy where orbitals along bonding axis are destabilized

UV Spectral Nomenclature

g-rays IR X-rays UV Microwave Radio Visible 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 g-rays X-rays UV IR Microwave Radio Visible

UV Spectroscopy Introduction The Spectroscopic Process 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

UV Spectroscopy Introduction 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)

Observed electronic transitions Here is a graphical representation UV Spectroscopy Introduction Observed electronic transitions Here is a graphical representation s* Unoccupied levels p* Atomic orbital Atomic orbital Energy n Occupied levels p s Molecular orbitals

Observed electronic transitions UV Spectroscopy Introduction Observed electronic transitions From the molecular orbital diagram, there are several possible electronic transitions that can occur, each of a different relative energy: s* s p n s* p* alkanes carbonyls unsaturated cmpds. O, N, S, halogens p* Energy n p s

s p n s* p* alkanes carbonyls unsaturated cmpds. O, N, S, halogens UV Spectroscopy Introduction 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 p n s* p* alkanes carbonyls unsaturated cmpds. O, N, S, halogens 150 nm 170 nm 180 nm √ - if conjugated! 190 nm 300 nm √

UV Spectroscopy Introduction Selection Rules Not all transitions that are possible are observed 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

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

E1 Energy E0 UV Spectroscopy Introduction Band Structure When these energy levels are superimposed, the effect can be readily explained – any transition has the possibility of being observed E1 Energy E0

Molecular UV-Visible Spectroscopy Molecular UV-Visible spectroscopy is driven by electronic absorption of UV-Vis radiation Molecular UV-Visible spectroscopy can: Enable structural analysis Detect molecular chromophores Analyze light-absorbing properties (e.g. for photochemistry) Nowdays, UV-Visible spectrometers are most commonly found in the diode array detectors (DAD) hooked to many LC systems. These are used for the study of LC eluents with chromophores. UV-Vis is not usually used for structural elucidation work given the power of NMR, MS and elemental methods. It is used for characterization purposes, for solid-state analysis, and for assessing photochemistry of compounds. For example, in pharmaceuticals, phototoxicity (photosafety) refers to the question of whether a drug will degrade in the presence of light, both in vivo and in a formulation. Photodegradation is particularly possible if the material has strong absorption between 290-700 nm and photostability studies are often conducted based on UV-Vis spectral data. Basic UV-Vis spectrophotometers acquire data in the 190-800 nm range and can be designed as “flow” systems. Figures from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv1

Molecular UV-Vis Spectroscopy: Terminology UV-Vis Terminology Chromophore: a UV-Visible absorbing functional group Bathochromic shift (red shift): to longer wavelengths Auxochrome: a substituent on a chromophore that causes a red shift Hypsochromic shift (blue shift): to shorter wavelengths Hyperchromic shift: to greater absorbance Hypochromic shift: to lesser absorbance