UV/Vis (Ultraviolet and Visible) Spectroscopy

Transcription

1 UV/Vis (Ultraviolet and Visible) Spectroscopy Agilent 8453 Diode Array UV-Vis Spectrophotometer Varian Cary 5000 UV-Vis-NIR Spectrophotometer

2 To Do s Read Chapters 13 & 14. Complete the end-of-chapter problems, 13-1, 13-2, 13-3,13-6 and Complete the end-of-chapter problems, 14-2, 14-5, 14-6, 14-7, 14-8, 14-9, 14-10, 14-12, 14-14, 14-16, and

3 Wavelength and Spectroscopy

4 What Molecular Changes Cause UV/Vis Absorptions? λ = 200 ~ 700 nm UV/Vis spectroscopy is used to detect the presence of certain functional groups called chromophores, especially in conjugate systemssuch as aromatics, dienes, polyenes, and α,β -unsaturated ketones Electronic excitation Absorption of UV or visible light occurs only when the energy of incident radiation is the same as that of an electronic transition in the molecule.

5 Electronic Transitions Only n π* and π π* transitions usually show up in nm. Only limited set of organic functional groups show UV/Vis absorptions.

6 Instrument (double beam) Typical λ = 200 ~ 700 nm

7 The Beer s Law I A λ = log ( ref ) I sample A λ = ε λ x c x l I ref I sample > 1, thus A λ I ref I sample I ref I sample is aways positive. = 1 A λ = 0 = 10 A λ = 1 ε λ : molar absorptivity constant or extinction coefficient (l mol -1 cm -1 ) c : concentration (mol/l) l : path length (cm) I ref I sample I ref I sample = 100 A λ = 2 = 1000 A λ = 3

8 Instrument (Diode Array) Rugged, fast, and reliable

9 Solvents and Cuvettes for UV/Vis Spectroscopy

10 Typical UV/Vis Spectra O CH 3

11 Franck-Condon Principle Nuclear motion is negligible during the time required for an electronic transition. A B Excitation can occur from the ground state to any of the excited state vibrational levels. The lines overlap and a continuous broad band is observed.

12 Difference Spectroscopy UV absorption spectra and temperature -difference spectra of HO5dCyt in 0.02 M NaOH/NaHCO3 buffer at ph 10.5 at 25 C (A, dashed line), 75 C (A, solid line) and C (B). Comparable spectra of dcyt in 0.02 M NaOH/NaHCO3 buffer at ph 7 at 25 C (C, dashed line), 75 C (C, solid line) and C (D).

13 Isosbestic Points ε HA = ε A- HA A - Isosbestic points are usually a diagnostic for the presence of only two absorbing species.

14 Spectral Shift blue shift red shift

15 Polymeric Nucleic Acids Have an Increased Molar Absorptivity When They are Converted to Smaller Units

16 Common Organic Chromophores conjugated and non-conjugated

17 The Carbonyl Groups λmax 270 ~ 300 nm εmax 10 ~ 100, n π* O C and R R 1 R O C H!* c o n The two orbitals, n and π*, are orthogonal to each other and promotion of electron requires significant changes in geometry. The electronic transition must be coupled with vibration that allows some overlap between these orbitals.

18 Other Carbonyl Groups! max " max!* O C H 3 C CH 3 H 3 C O C Cl (hexane) (hexane) R C O X n O C H 3 C NH 2 (water) O!* H 3 C C OEt (water) C=O n (X) H 3 C O C OH (ethanolr) n

19 Different Carbonyl Compounds and Their UV/Vis

20 Solvent Effects C OI H O!* R n

21 Aldehydes and Ketons

22 Unconjugated Alkenes

23 Conjugated Alkenes

24 Conjugation and Spectral Shifts

25 β-carotene

26 Diene Isomers λmax = 239 nm PPM λmax = 278 nm PPM

27 Empirical Calculation of λmax for Conjugated Alkenes

28 Empirical Calculation - Examples

29 Empirical Calculation More Examples

30 Steric Hindrance and Spectral Shifts H 3 C CH 3 CH 3 CH 3 CH 3 CH 3 H 3 C CH 2 H 3 C CH 2! max " max ,000 H 3 C CH 3 CH 3 CH 3 Twisted diene low conjugation λmax and εmax H 3 C CH 2

31 Conjugated Carbonyls

32 Empirical Calculation alkyl or ring residues

33 Empirical Calculation - Examples

34 Exocyclic Alkene Issue O β-alkyl 3 x 18 δ+ alkyl 2 x 5 exocyclic C=C 2 x 30 extra conjugation 351 nm Observed 348 nm

35 Homoconjugation

36 Homoconjugation- More Examples

37 Solvent Effect In hexane λmax = 295 εmax = 50 In ethanol λmax = 255 εmax = 12, (β-alkyl) 30 (β-oh) 257nm

38 Charge Transfer(CT) Bands A + D A D h! A D CT complex

39 Intramolecular CT Bands O!-!+

40 Benzene Chromophore O H n π*

41 Solvent Effects

42 Benzene Bands Designation

43 Substituent Effects

44 Benzene Derivatives - Monosubstituted

45 ph Sensitive Derivatives O OH OH - O O OH OH - O

46 ph Titration of Phenol Isosbestic point

47 pka Measurement

48 Aromatic Amino Acids H 3 N R O O R = Phe OH Tyr H N Trp

49 Benzene Derivatives - Disubstituted The mono-substituted moiety with the largest red shift will dominate the absorption of a di-substituted benzene.

50 Para-substituted Benzenes If one substituent is e-withdrawing and the other is e-donating, a significant red-shift is observed due to the extended conjugation.

51 Disubstituted Anilines

52 Disubstituted Benzaldehydes

53 Disubstituted Benzoic Acids

54 Fused Ring Systems

55 Heterocyclic Aromatics

56 Steric Effect Cis & Trans

57 Steric Effect E & Z E E Z E Z Z! max " max 56,200 30,600 29,500

58 Steric Effect - Biphenyls

59 Empirical Calculation Benzoyl Derivatives

60 Empirical Calculation Examples Br 246 (ketone) + 3 (o-ring) + 2 (m-br) HO CO 2 H 230 (acid) + 7 x 2 (m-oh) + 25 (p-oh) O! max = 251nm obs = 253 nm HO OH! max = 269nm obs = 270 nm

61 Tautomerization O OH 239 (14,100) N H N N OH 260 (2,200) N 257 (2,750)

62 Porphyrin Chromophore

63 Exciton Coupling

64 UV/Vis Analysis A single band (e = 10 ~ 100) in 250 ~ 300 nm, no major absorption in 200 ~ 250 nm. n π*, C=O (ketone and aldehyde), C=N. N=N, -NO2 etc Two bands (e = 1,000 ~ 10,000) > 200 nm. π π*, aromatics Bands (e = 10,000 ~ 20,000) > 210 nm. π π*, conjugated C=C and C=O Compounds that are highly colored A long-chain conjugated system A polycyclic aromatic system ph-depended absorption Ionizable group attached to chromophore