1. Absorption Spectroscopy of Biopolymers
Overview

4. Visible & near-UV region wavelength (nm)
Microwave & radiowave region frequency (Hz)
Infared region wavenumber (cm-1)
Far-UV, x-ray, g-ray energy (DE=hn)

5. Absorption & Emission
Rapid process(10-15s)

6. Absorption & Emission

7. Radiation-Induced Transition
Absorption
Stimulated emission
Spontaneous emission

8. UV-Visible Spectroscopy
Ultraviolet-visible spectroscopy involves the absorption of ultraviolet/visible light by a molecule causing the promotion of an electron from a ground electronic state to an excited electronic state.
Ultraviolet/Visible light:
wavelengths (l) between 190 and 800 nm

9. UV-visible spectrum The two main properties of an absorbance peak are:
Absorption wavelength
lmax
Absorption intensity
Amax
Housecroft and Sharpe, p. 466

10. Beer-Lambert Law e = A/cb A = ebc A = ec (when b is 1 cm)
log(I0/I) = ebc
e = A/cb
A = ebc
A = ec (when b is 1 cm)
I0 = intensity of incident light
I = intensity of transmitted light
= molar absoptivity coefficient in cm2 mol-1
c = concentration in mol L-1
b = pathlength of absorbing solution in cm-1
A = absorbance = log(Io/I) ℓ
0.1 cm

11. Beer-Lambert Law A Absorbance or optical density (OD)
e absorptivity; M-1 cm-1
c concentration; M
T transmittance

12. Transmittance, Absorbance, and Cell Pathlength

13. Deviations from the Beer-Lambert Law
Low c
High c
The Beer-Lambert law assumes that all molecules contribute to the absorption and that no absorbing molecule is in the shadow of another

14. Sample Concentrations
Solution too concentrated Diluted five-fold

15. Molar absorptivities (e)
Molar absoptivities are very large for strongly absorbing chromophores (e >10,000) and very small if the absorption is weak (e = 10 to 100). The magnitude of e reflects both the size of the chromophore and the probability that light of a given wavelength will be absorbed when it strikes the chromophore. A general equation stating this relationship may be written as follows:
= 0.87 x 1020P x a
where P is the transition probability (0 to 1)
a is the chromophore area in cm2
The transition probability depends on a number of factors including where the transition is an “allowed” transition or a “forbidden” transition

16. UV-visible spectrum of 4-nitroanaline
Molecular mass = 138
Solvent: Ethanol
Concentration: mg L-1
Pathlength: 1 cm
Harwood and Claridge, p. 18

17. UV-visible spectrum of 4-nitroanaline
Determine the absorption maxima (lmax) and absorption intensities (A) from the spectrum:
lmax = 227 nm, A227 = 1.55 lmax = 375 nm, A375 = 1.75
2. Calculate the concentration of the compound:
(1.54 x 10-2 g L-1)/(138 g/mol) = 1.12 x 10-4 mol L-1
Determine the molar absorptivity coefficients (e) from the Beer-Lambert Law: e = A/cℓ
e227 = 1.55/(1.0 cm x 1.12 x 10-4 mol L-1) = 13,900 mol-1 L cm-1
e375 = 1.75/(1.0 cm x 1.12 x 10-4 mol L-1) = 15,700 mol-1 L cm-1

18. UV-visible spectroscopy definitions
chromophore Any group of atoms that absorbs light whether or not a color is thereby produced.
auxochrome A group which extends the conjugation of a chromophore by sharing of nonbonding electrons.
bathochromic shift The shift of absorption to a longer wavelength.
hypsochromic shift The shift of absorption to a shorter wavelength.
hyperchromic effect An increase in absorption intensity.
hypochromic effect A decrease in absorption intensity.

19. Absorption and Emission of Photons

20. Absorption and Emission
Absorption: A transition from a lower level to a higher level with transfer of energy from the radiation field to an absorber, atom, molecule, or solid.
Emission: A transition from a higher level to a lower level with transfer of energy from the emitter to the radiation field. If no radiation is emitted, the transition from higher to lower energy levels is called nonradiative decay.

21. Singlet and Triplet Excited States

22. Absorption and emission pathways
McGarvey and Gaillard, Basic Photochemistry at

23. Selection Rules
In electronic spectroscopy there are three selection rules which determine whether or not transitions are formally allowed:
Spin selection rule: DS = 0
allowed transitions: singlet  singlet or triplet  triplet forbidden transitions: singlet  triplet or triplet  singlet
Changes in spin multiplicity are forbidden

24. Selection rules
Laporte selection rule: there must be a change in the parity (symmetry) of the complex
Laporte-allowed transitions: g  u Laporte-forbidden transitions: g  g or u  u
g stands for gerade – compound with a center of symmetry
u stands for ungerade – compound without a center of symmetry
Selection rule of Dℓ = ± 1 (ℓ is the azimuthal or orbital quantum number, where ℓ = 0 (s orbital), 1 (p orbital), 2 (d orbital), etc.)
allowed transitions: s  p, p  d, d  f, etc.
forbidden transitions: s  s, d  d, p  f, etc.

26. s and s* orbitals

27. p and p* orbitals

28. Electronic Transitions: p  p*
/Spectrpy/UV-Vis/uvspec.htm#uv2
The p  p* transition involves orbitals that have significant overlap, and the probability is near 1.0 as they are “symmetry allowed”.
McGarvey and Gaillard, Basic Photochemistry at

29. p  p* transitions - Triple bonds
Organic compounds with -C≡C- or -C≡N groups, or transition metals complexed by C≡N- or C≡O ligands, usually have “low-lying” p* orbitals

30. Electronic Transitions: n  p*
/Spectrpy/UV-Vis/uvspec.htm#uv2
The n-orbitals do not overlap at all well with the p* orbital, so the probability of this excitation is small. The e of the np* transition is about 103 times smaller than e for the pp* transition as it is “symmetry forbidden”.
McGarvey and Gaillard, Basic Photochemistry at

32. Lycopene from Tomatoes

33. Chlorophyll
B-carotene
hemoglobin

34. Quantitative Analysis
A plot of absorption versus wavelength is the absorption spectrum

35. Solutions containing the amino acids tryptophan and tyrosine can be analyzed under alkaline conditions (0.1 M KOH) from their different uv spectra. The extinction coefficients under these conditions at 240 nm and 280 nm are
A 10-mg smaple of the protein glucagon is hydrolyzed to its constituent amino acids and diluter to 100 mL in 0.1 M KOH. The absorbance of this solution (1 cm path) was at 240 nm and at 280 nm. Estimate the content of tryptophan and tyrosine in mol (g protein)-1

36. Isosbestic points Isosbestic wavelength
the wavelength at which two or more components have the same extinction coefficient The occurrence of two or more isosbestics in the spectra of a series of solutions of the same total concentration demonstrates the presence of two and only two components absorbing in that spectra region.

37. Isosbestic points

38. UV spectrum of DNA from E. coli
UV spectrum of BSA
UV spectrum of DNA from E. coli

39. UV Absorption of amino acid

40. Effect of Secondary structure

41. Origin of Spectroscopic Changes
Change in local charge distribution
Change in dielectric constant
Change in bonding interaction
Change in dynamic coupling between different parts of the molecule

44. Light sensitive protein
Human Eye
Retina
Light sensitive protein
Retina
Outer segment

47. Rhodopsin is a protein in the membrane of the photoreceptor cell in the retina of the eye. It catalyses the only light sensitive step in vision. The 11-cis-retinal chromophore lies in a pocket of the protein and is isomerised to all-trans retinal when light is absorbed. The isomerisation of retinal leads to a change of the shape of rhodopsin which triggers a cascade of reactions which lead to a nerve impulse which is transmitted to the brain by the optical nerve
1BRD

48. 1BRD
1BM1