5 Spectroscopic Techniques and Common Uses Spectroscopic Techniques and Common UsesUV-visUV-vis regionQuantitative analysis/Beer’s LawAtomic AbsorptionQuantitative analysisBeer’s LawFT-IRIR/MicrowaveFunctional Group AnalysisRamanIR/UVFunctional Group Analysis/quantFT-NMRRadio wavesStructure determinationX-Ray SpectroscopyX-raysElemental AnalysisX-ray Crystallography3-D structure Anaylysis
6 Different Spectroscopies UV-vis – electronic states of valence e/d-orbital transitions for solvated transition metalsFluorescence – emission of UV/vis by certain moleculesFT-IR – vibrational transitions of moleculesFT-NMR – nuclear spin transitionsX-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 UVUV absorbance is about 1000 x easier to detect per mole than NMRStill 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 detectorsOne wavelength may not be the best for all compound in a mixture.Affects quantitative interpretation of HPLC peak heights
8 Uses for UV, continuedKnowing UV can help you know when to be skeptical of quant results. Need to calibrate response factorsAssessing 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 sensitivee.g. validation of cleaning procedure for a production vesselBut 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 enoneπnπ*-*; max=218=11,000n-*; max=320=100What 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 photonLUMO g antibonding molecular orbitalHOMO u bonding molecular orbital
11 How Do UV spectrometers work? Rotates, to achieve scanMatched quartz cuvettesSample in solution at ca M.System protects PM tube from stray lightD2 lamp-UVTungsten lamp-VisDouble Beam makes it a difference techniqueTwo photomultiplier inputs, differential voltage drives amplifier.
12 Diode Array DetectorsDiode array alternative puts grating, array of photosens. Semiconductors after the light goes through the sample. Advantage, speed, sensitivity,The Multiplex advantageDisadvantage, resolution is 1 nm, vs 0.1 nm for normal UVModel 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 absorbersJust heteroatoms are not enough but C=O are reliableMost 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, inorganicsSolvent 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 knownUV bands are much broader than the photonic transition event. This is because vibration levels are superimposed on UV.AbsorbanceWavelength, , generally in nanometers (nm)0.04008001.0200maxwith certain extinction UVVisible
15 Solvents for UV (showing high energy cutoffs) Water 205CH3CN 210C6HEther 210EtOH 210Hexane 210MeOH 210Dioxane 220THF 220CH2Cl2 235CHCl3 245CCl4 265benzene 280Acetone 300Various buffers for HPLC, check before using.
16 Organic compounds (many of them) have UV spectra One thing is clearUvs can be very non-specificIts hard to interpret except at a cursory level, and to say that the spectrum is consistent with the structureEach band can be a superposition of many transitionsGenerally 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 103Antilog of 3.86
18 Can we calculate UVs?Semi-empirical (MOPAC) at AM1, then ZINDO for config. interaction level 14Bandwidth set to 3200 cm-1
19 The orbitals involvedShowing atoms whose MO’s contribute most to the bands
20 The Quantitative Picture (power in)P(power out)Transmittance:T = P/P0Absorbance:A = -log10 T = log10 P0/PB(path through sample)The Beer-Lambert Law (a.k.a. Beer’s Law):A = ebcWhere the absorbance A has no units, since A = log10 P0 / Pe is the molar absorbtivity with units of L mol-1 cm-1b is the path length of the sample in cmc is the concentration of the compound in solution, expressed in mol L-1 (or M, molarity)
21 Beer-Lambert LawLinear absorbance with increased concentration--directly proportionalMakes UV useful for quantitative analysis and in HPLC detectorsAbove 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 LawAl1 = el1bce is molar absorptivity (unique for a given compound at l1)b is path lengthc concentration
23 Beer’s Law A = -logT = log(P0/P) = ebc T = Psolution/Psolvent = P/P0 cuvettesourceslitdetectorA = -logT = log(P0/P) = ebcT = Psolution/Psolvent = P/P0Works for monochromatic lightCompound x has a unique e at different wavelengths
24 Characteristics of Beer’s Law Plots One wavelengthGood plots have a range of absorbances from to 1.000Absorbances over are not that valid and should be avoided2 orders of magnitude
25 Standard Practice Prepare standards of known concentration Measure absorbance at lmaxPlot A vs. concentrationObtain slopeUse slope (and intercept) to determine the concentration of the analyte in the unknown
27 UV-Vis Spectroscopy UV- organic molecules Outer electron bonding transitionsconjugationVisible – metal/ligands in solutiond-orbital transitionsInstrumentation
28 Characteristics of UV-Vis spectra of Organic Molecules Absorb mostly in UV unless highly conjugatedSpectra are broad, usually to broad for qualitative identification purposesExcellent for quantitative Beer’s Law-type analysesThe most common detector for an HPLC
29 } = hv Molecules have quantized energy levels: ex. electronic energy levels.hv}energyenergy= hvQ: 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
33 max = 135 nm (a high energy transition) Ethanemax = 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.
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 ; = 1860n max = 279 nm ; = 15
40 Polyenes, and Unsaturated Carbonyl groups; an Empirical triumph R.B. Woodward, L.F. Fieser and othersPredict max for π* in extended conjugation systems to within ca. 2-3 nm.Attached group increment, nmExtend conjugation +30Addn exocyclic DB +5Alkyl +5O-Acyl 0S-alkyl +30O-alkyl +6NR2 +60Cl, Br +5Homoannular, base 253 nmAcyclic, base 217 nmheteroannular, base 214 nm
41 Similar for Enones O x b b g d,+ 202 227 239 215 Base Values, add these increments…bgd,+X=H 207X=R 215X=OH 193X=OR 193Extnd C=C+30Add exocyclic C=C+5Homoannular diene+39alkyl+10+12+18OH+35+50OAcyl+6O-alkyl+17+31NR2S-alkylCl/Br+15/+25+12/+30With solvent correction of…..WaterEtOHCHClDioxaneEt2OHydrcrbn -11
42 Some Worked ExamplesBase value x alkyl subst exo DB total 232 Obs. 237Base value x alkyl subst exo DB total 234 Obs. 235Base value ß alkyl subst total 239 Obs. 237
43 Distinguish Isomers!Base value x alkyl subst exo DB total 239 Obs. 238Base value x alkyl subst total 273 Obs. 273
44 Generally, extending conjugation leads to red shift “particle in a box” QM theory; bigger boxSubstituents 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 orbitalsOne 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 bandAlkyl208(7800)260(220)---OH211(6200)270(1450)-O-236(9400)287(2600)-OCH3217(6400)269(1500)NH2230(8600)280(1400)-F204(6200)254(900)-Cl210(7500)257(170)-Br-I207(7000)258/285(610/180)-NH3+203(7500)254(160)-C=CH2248(15000)282(740)-CCH248(17000)278(6500-C6H6250(14000)-C(=O)H242(14000)328(55)-C(=O)R238(13000)276(800)320(40)-CO2H226(9800)272(850)-CO2-224(8700)268(800)-CN224(13000)271(1000)-NO2252(10000)280(1000)330(140)Benzenoid aromaticsUV of Benzene in heptaneFrom Crewes, Rodriguez, Jaspars, Organic Structure Analysis
47 Substituent effects don’t really add up Can’t tell any thing about substitution geometryException 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 acidIn p-hydroxybenzoic acid, it is at the phenol or benzoic acid frequency
48 HeterocyclesNitrogen 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 titrationsExample here gives detn of endpoint for bromcresol greenBinding studiesForm I to form IIIsosbestic pointsSingle clear point, can exclude intermediate state, exclude light scattering and Beer’s law appliesBinding 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 multiplicityPhosphorescence: absorption of radiation to an excited state, followed by emission of radiation to a lower state of different multiplicitySinglet 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
54 Fixed Wavelength Instrument LED serve as sourcePseudo-monochromatic light sourceNo monochrometer necessary/ wavelength selection occurs by turning on the appropriate LED4 LEDs to choose fromsamplebeam of lightLEDsphotodyode
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 holderVisible; can be plastic or glassUV; you must use quartz
61 Advantages/disadvantages Scanning instrumentHigh spectral resolution (63000), l/DlLong data acquisition time (several minutes)Low throughputDiode arrayFast acquisition time (a couple of seconds), compatible with on-line separationsHigh throughput (no slits)Low resolution (2 nm)