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Light interacting with matter as an analytical tool

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1 Light interacting with matter as an analytical tool
UV-VIS SPECTROSCOPY Light interacting with matter as an analytical tool

2 Electronic Excitation by UV/Vis Spectroscopy :
UV: valance electronic excitation Radio waves: Nuclear spin states (in a magnetic field) IR: molecular vibrations X-ray: core electron excitation

3 Where in the spectrum are these transitions?

4 Spectroscopic Techniques and
Spectroscopic Techniques and Chemistry they Probe UV-vis UV-vis region bonding electrons Atomic Absorption atomic transitions (val. e-) FT-IR IR/Microwave vibrations, rotations Raman IR/UV vibrations FT-NMR Radio waves nuclear spin states X-Ray Spectroscopy X-rays inner electrons, elemental X-ray Crystallography 3-D structure

5 Spectroscopic Techniques and Common Uses
Spectroscopic Techniques and Common Uses UV-vis UV-vis region Quantitative analysis/Beer’s Law Atomic Absorption Quantitative analysis Beer’s Law FT-IR IR/Microwave Functional Group Analysis Raman IR/UV Functional Group Analysis/quant FT-NMR Radio waves Structure determination X-Ray Spectroscopy X-rays Elemental Analysis X-ray Crystallography 3-D structure Anaylysis

6 Different Spectroscopies
UV-vis – electronic states of valence e/d-orbital transitions for solvated transition metals Fluorescence – emission of UV/vis by certain molecules FT-IR – vibrational transitions of molecules FT-NMR – nuclear spin transitions X-Ray Spectroscopy – electronic transitions of core electrons

7 Why should we learn this stuff
Why should we learn this stuff? After all, nobody solves structures with UV any longer! Many organic molecules have chromophores that absorb UV UV absorbance is about 1000 x easier to detect per mole than NMR Still used in following reactions where the chromophore changes. Useful because timescale is so fast, and sensitivity so high. Kinetics, esp. in biochemistry, enzymology. Most quantitative Analytical chemistry in organic chemistry is conducted using HPLC with UV detectors One wavelength may not be the best for all compound in a mixture. Affects quantitative interpretation of HPLC peak heights

8 Uses for UV, continued Knowing UV can help you know when to be skeptical of quant results. Need to calibrate response factors Assessing purity of a major peak in HPLC is improved by “diode array” data, taking UV spectra at time points across a peak. Any differences could suggest a unresolved component. “Peak Homogeneity” is key for purity analysis. Sensitivity makes HPLC sensitive e.g. validation of cleaning procedure for a production vessel But you would need to know what compounds could and could not be detected by UV detector! (Structure!!!) One of the best ways for identifying the presence of acidic or basic groups, due to big shifts in  for a chromophore containing a phenol, carboxylic acid, etc. “hypsochromic” shift “bathochromic” shift

9 The UV Absorption process
  * 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 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 = nm. Any of these require that incoming photons match in energy the gap corrresponding to a transition from ground to excited state. Energies correspond to a 1-photon of 300 nm light are ca. 95 kcal/mol

10 What are the nature of these absorptions?
Example for a simple enone π n π* -*; max=218 =11,000 n-*; max=320 =100 What are the nature of these absorptions? Example:   * transitions responsible for ethylene UV absorption at ~170 nm calculated with ZINDO semi-empirical excited-states methods (Gaussian 03W): h 170nm photon LUMO g antibonding molecular orbital HOMO u bonding molecular orbital

11 How Do UV spectrometers work?
Rotates, to achieve scan Matched quartz cuvettes Sample in solution at ca M. System protects PM tube from stray light D2 lamp-UV Tungsten lamp-Vis Double Beam makes it a difference technique Two photomultiplier inputs, differential voltage drives amplifier.

12 Diode Array Detectors Diode array alternative puts grating, array of photosens. Semiconductors after the light goes through the sample. Advantage, speed, sensitivity, The Multiplex advantage Disadvantage, resolution is 1 nm, vs 0.1 nm for normal UV Model from Agilent literature. Imagine replacing “cell” with a microflow cell for HPLC!

13 Experimental details What compounds show UV spectra?
Generally think of any unsaturated compounds as good candidates. Conjugated double bonds are strong absorbers Just heteroatoms are not enough but C=O are reliable Most compounds have “end absorbance” at lower frequency. Unfortunately solvent cutoffs preclude observation. You will find molar absorbtivities  in L•cm/mol, tabulated. Transition metal complexes, inorganics Solvent must be UV grade (great sensitivity to impurities with double bonds) The NIST databases have UV spectra for many compounds

14 An Electronic Spectrum
Make solution of concentration low enough that A≤ 1 (Ensures Linear Beer’s law behavior) Even though a dual beam goes through a solvent blank, choose solvents that are UV transparent. Can extract the  value if conc. (M) and b (cm) are known UV bands are much broader than the photonic transition event. This is because vibration levels are superimposed on UV. Absorbance Wavelength, , generally in nanometers (nm) 0.0 400 800 1.0 200 maxwith certain extinction  UV Visible

15 Solvents for UV (showing high energy cutoffs)
Water 205 CH3CN 210 C6H Ether 210 EtOH 210 Hexane 210 MeOH 210 Dioxane 220 THF 220 CH2Cl2 235 CHCl3 245 CCl4 265 benzene 280 Acetone 300 Various buffers for HPLC, check before using.

16 Organic compounds (many of them) have UV spectra
One thing is clear Uvs can be very non-specific Its hard to interpret except at a cursory level, and to say that the spectrum is consistent with the structure Each band can be a superposition of many transitions Generally we don’t assign the particular transitions. From Skoog and West et al. Ch 14

17 An Example--Pulegone Frequently plotted as log of molar extinction 
So at 240 nm, pulegone has a molar extinction of 7.24 x 103 Antilog of 3.86

18 Can we calculate UVs? Semi-empirical (MOPAC) at AM1, then ZINDO for config. interaction level 14 Bandwidth set to 3200 cm-1

19 The orbitals involved Showing atoms whose MO’s contribute most to the bands

20 The Quantitative Picture
(power in) P (power out) Transmittance: T = P/P0 Absorbance: A = -log10 T = log10 P0/P B(path through sample) The Beer-Lambert Law (a.k.a. Beer’s Law): A = ebc Where the absorbance A has no units, since A = log10 P0 / P e is the molar absorbtivity with units of L mol-1 cm-1 b is the path length of the sample in cm c is the concentration of the compound in solution, expressed in mol L-1 (or M, molarity)

21 Beer-Lambert Law Linear absorbance with increased concentration--directly proportional Makes UV useful for quantitative analysis and in HPLC detectors Above a certain concentration the linearity curves down, loses direct proportionality--Due to molecular associations at higher concentrations. Must demonstrate linearity in validating response in an analytical procedure.

22 Quantitative Spectroscopy
Beer’s Law Al1 = el1bc e is molar absorptivity (unique for a given compound at l1) b is path length c concentration

23 Beer’s Law A = -logT = log(P0/P) = ebc T = Psolution/Psolvent = P/P0
cuvette source slit detector A = -logT = log(P0/P) = ebc T = Psolution/Psolvent = P/P0 Works for monochromatic light Compound x has a unique e at different wavelengths

24 Characteristics of Beer’s Law Plots
One wavelength Good plots have a range of absorbances from to 1.000 Absorbances over are not that valid and should be avoided 2 orders of magnitude

25 Standard Practice Prepare standards of known concentration
Measure absorbance at lmax Plot A vs. concentration Obtain slope Use slope (and intercept) to determine the concentration of the analyte in the unknown

26 Typical Beer’s Law Plot

27 UV-Vis Spectroscopy UV- organic molecules
Outer electron bonding transitions conjugation Visible – metal/ligands in solution d-orbital transitions Instrumentation

28 Characteristics of UV-Vis spectra of Organic Molecules
Absorb mostly in UV unless highly conjugated Spectra are broad, usually to broad for qualitative identification purposes Excellent for quantitative Beer’s Law-type analyses The most common detector for an HPLC

29 }  = hv Molecules have quantized energy levels:
ex. electronic energy levels. hv } energy energy  = hv Q: Where do these quantized energy levels come from? A: The electronic configurations associated with bonding. Each electronic energy level (configuration) has associated with it the many vibrational energy levels we examined with IR.

30 Broad spectra Overlapping vibrational and rotational peaks
Solvent effects

31 Molecular Orbital Theory
Fig 18-10

32 s* p* 2p 2p n p s s* 2s 2s s

33 max = 135 nm (a high energy transition)
Ethane max = 135 nm (a high energy transition) Absorptions having max < 200 nm are difficult to observe because everything (including quartz glass and air) absorbs in this spectral region.

34 = hv =hc/ Example: ethylene absorbs at longer wavelengths:
max = 165 nm = 10,000

35 The n to pi* transition is at even lower wavelengths but is not as strong as pi to pi* transitions. It is said to be “forbidden.” Example: Acetone: n max = 188 nm ; = 1860 n max = 279 nm ; = 15

36  135 nm  165 nm n 183 nm weak  150 nm

37 1,3 butadiene: max = 217 nm ; = 21,000
Conjugated systems: Preferred transition is between Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO). Note: Additional conjugation (double bonds) lowers the HOMO-LUMO energy gap: Example: 1,3 butadiene: max = 217 nm ; = 21,000 1,3,5-hexatriene max = 258 nm ; = 35,000

38 Similar structures have similar UV spectra:
max = 240, 311 nm max = 238, 305 nm max = 173, 192 nm

39 max = (8) + 11*( *11) = 476 nm max(Actual) = 474.

40 Polyenes, and Unsaturated Carbonyl groups; an Empirical triumph
R.B. Woodward, L.F. Fieser and others Predict max for π* in extended conjugation systems to within ca. 2-3 nm. Attached group increment, nm Extend conjugation +30 Addn exocyclic DB +5 Alkyl +5 O-Acyl 0 S-alkyl +30 O-alkyl +6 NR2 +60 Cl, Br +5 Homoannular, base 253 nm Acyclic, base 217 nm heteroannular, base 214 nm

41 Similar for Enones O x  b  b g d,+ 202 227 239 215
Base Values, add these increments… b g d,+ X=H 207 X=R 215 X=OH 193 X=OR 193 Extnd C=C +30 Add exocyclic C=C +5 Homoannular diene +39 alkyl +10 +12 +18 OH +35 +50 OAcyl +6 O-alkyl +17 +31 NR2 S-alkyl Cl/Br +15/+25 +12/+30 With solvent correction of….. Water EtOH CHCl Dioxane Et2O Hydrcrbn -11

42 Some Worked Examples Base value x alkyl subst exo DB total 232 Obs. 237 Base value x alkyl subst exo DB total 234 Obs. 235 Base value ß alkyl subst total 239 Obs. 237

43 Distinguish Isomers! Base value x alkyl subst exo DB total 239 Obs. 238 Base value x alkyl subst total 273 Obs. 273

44 Generally, extending conjugation leads to red shift
“particle in a box” QM theory; bigger box Substituents attached to a chromophore that cause a red shift are called “auxochromes” Strain has an effect… max

45 Interpretation of UV-Visible Spectra
Transition metal complexes; d, f electrons. Lanthanide complexes – sharp lines caused by “screening” of the f electrons by other orbitals One advantage of this is the use of holmium oxide filters (sharp lines) for wavelength calibration of UV spectrometers. See Shriver et al. Inorganic Chemistry, 2nd Ed. Ch. 14

46 Benzenoid aromatics UV of Benzene in heptane Group K band ()
B band() R band Alkyl 208(7800) 260(220) -- -OH 211(6200) 270(1450) -O- 236(9400) 287(2600) -OCH3 217(6400) 269(1500) NH2 230(8600) 280(1400) -F 204(6200) 254(900) -Cl 210(7500) 257(170) -Br -I 207(7000) 258/285(610/180) -NH3+ 203(7500) 254(160) -C=CH2 248(15000) 282(740) -CCH 248(17000) 278(6500 -C6H6 250(14000) -C(=O)H 242(14000) 328(55) -C(=O)R 238(13000) 276(800) 320(40) -CO2H 226(9800) 272(850) -CO2- 224(8700) 268(800) -CN 224(13000) 271(1000) -NO2 252(10000) 280(1000) 330(140) Benzenoid aromatics UV of Benzene in heptane From Crewes, Rodriguez, Jaspars, Organic Structure Analysis

47 Substituent effects don’t really add up
Can’t tell any thing about substitution geometry Exception to this is when adjacent substituents can interact, e.g hydrogen bonding. E.g the secondary benzene band at 254 shifts to 303 in salicylic acid In p-hydroxybenzoic acid, it is at the phenol or benzoic acid frequency

48 Heterocycles Nitrogen heterocycles are pretty similar to the benzenoid anaologs that are isoelectronic. Can study protonation, complex formation (charge transfer bands)

49 Quantitative analysis
Great for non-aqueous titrations Example here gives detn of endpoint for bromcresol green Binding studies Form I to form II Isosbestic points Single clear point, can exclude intermediate state, exclude light scattering and Beer’s law applies Binding of a lanthanide complex to an oligonucleotide

50 More Complex Electronic Processes
Fluorescence: absorption of radiation to an excited state, followed by emission of radiation to a lower state of the same multiplicity Phosphorescence: absorption of radiation to an excited state, followed by emission of radiation to a lower state of different multiplicity Singlet state: spins are paired, no net angular momentum (and no net magnetic field) Triplet state: spins are unpaired, net angular momentum (and net magnetic field) Fluorescence is a process that involves a singlet to singlet transition. Phosphorescence is a process that involves a triplet to singlet transition

51 Metal ion transitions DE Degenerate D-orbitals D-orbitals of naked Co
of hydrated Co2+ Octahedral Configuration

52 Octahedral Geometry H2O H2O H2O Co2+ H2O H2O H2O

53 Instrumentation Fixed wavelength instruments Scanning instruments
Diode Array Instruments

54 Fixed Wavelength Instrument
LED serve as source Pseudo-monochromatic light source No monochrometer necessary/ wavelength selection occurs by turning on the appropriate LED 4 LEDs to choose from sample beam of light LEDs photodyode

55 Scanning Instrument Scanning Instrument monochromator slit Tungsten
Filament (vis) Photomultiplier tube slit cuvette Deuterium lamp Filament (UV)

56 sources Tungten lamp (350-2500 nm) Deuterium (200-400 nm)
Xenon Arc lamps ( nm)

57 Monochromator Braggs law, nl = d(sin i + sin r)
Angular dispersion, dr/dl = n / d(cos r) Resolution, R = l/Dl = nN, resolution is extended by concave mirrors to refocus the divergent beam at the exit slit

58 Sample holder Visible; can be plastic or glass UV; you must use quartz

59 Single beam vs. double beam
Source flicker

60 Diode array Instrument
mirror Diode array detector 328 individual detectors Tungsten Filament (vis) slit slit cuvette Deuterium lamp Filament (UV) monochromator

61 Advantages/disadvantages
Scanning instrument High spectral resolution (63000), l/Dl Long data acquisition time (several minutes) Low throughput Diode array Fast acquisition time (a couple of seconds), compatible with on-line separations High throughput (no slits) Low resolution (2 nm)

62 HPLC-UV HPLC Pump HPLC column 6-port valve Mobile phase Sample loop
UV detector syringe Solvent waste

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