Overview

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

Rapid process(10 -15 s)

I
 
 c n
  
• Absorption
• Stimulated emission
• Spontaneous emission

• 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

UV-visible spectrum
The two main properties of an absorbance peak are:
1. Absorption wavelength l max
2. Absorption intensity
A max
Housecroft and Sharpe, p. 466

Beer-Lambert Law
:
Beer-Lambert Law log( I
0
/ I ) = e b c e
= A/ c b
A = e bc
A = e c
(when b is 1 cm)
I
0
= intensity of incident light
I = intensity of transmitted light e
= molar absoptivity coefficient in cm 2 mol -1 c = concentration in mol L -1 b = pathlength of absorbing solution in cm -1
A = absorbance = log( I o
/ I )
0.1 cm ℓ http://www.hellma-worldwide.de/en/default.asp

A
 log
I
0
I t
 e bl
  log T
•
• A Absorbance or optical density (OD) e absorptivity; M -1 cm -1
• c concentration; M
• T transmittance

Transmittance, Absorbance, and Cell
Pathlength http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm

Low c
Deviations from the Beer-Lambert Law
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 http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm

Sample Concentrations
Solution too concentrated Diluted five-fold

UV-visible spectrum of 4-nitroanaline
NH
2
NO
2
Molecular mass = 138
Solvent: Ethanol
Concentration: 15.4 mg L -1
Pathlength: 1 cm
Harwood and Claridge, p. 18

UV-visible spectrum of 4-nitroanaline
1. Determine the absorption maxima ( l max
) and absorption intensities
( A ) from the spectrum: l max
= 227 nm, A
227
= 1.55
l max
= 375 nm, A
375
= 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
3. Determine the molar absorptivity coefficients ( e
) from the Beer-
Lambert Law: e
= A / c ℓ e
227
= 1.55/(1.0 cm x 1.12 x 10 -4 mol L -1 ) = 13,900 mol -1 L cm -1 e
375
= 1.75/(1.0 cm x 1.12 x 10 -4 mol L -1 ) = 15,700 mol -1 L cm -1

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: e
= 0.87 x 10 20 P x a where P is the transition probability (0 to 1) a is the chromophore area in cm 2
The transition probability depends on a number of factors including where the transition is an “allowed” transition or a “forbidden” transition http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv2

UV-visible spectrum of 4-nitroanaline
NH
2
NO
2
Molecular mass = 138
Solvent: Ethanol
Concentration: 15.4 mg L -1
Pathlength: 1 cm
Harwood and Claridge, p. 18

UV-visible spectrum of 4-nitroanaline
1. Determine the absorption maxima ( l max
) and absorption intensities
( A ) from the spectrum: l max
= 227 nm, A
227
= 1.55
l max
= 375 nm, A
375
= 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
3. Determine the molar absorptivity coefficients ( e
) from the Beer-
Lambert Law: e
= A / c ℓ e
227
= 1.55/(1.0 cm x 1.12 x 10 -4 mol L -1 ) = 13,900 mol -1 L cm -1 e
375
= 1.75/(1.0 cm x 1.12 x 10 -4 mol L -1 ) = 15,700 mol -1 L cm -1

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.

Absorption and Emission of Photons http://micro.magnet.fsu.edu/optics/lightandcolor/frequency.html

Absorption and Emission
Absorption
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.
http://www.chemistry.vt.edu/chem-ed/spec/spectros.html

Singlet and Triplet Excited States http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/lumin1.htm

Absorption and emission pathways
McGarvey and Gaillard, Basic Photochemistry at http://classes.kumc.edu/grants/dpc/instruct/index2.htm

Selection Rules
In electronic spectroscopy there are three selection rules which determine whether or not transitions are formally allowed:
1. Spin selection rule:
D
S = 0 allowed transitions: singlet
 singlet or triplet
 triplet forbidden transitions: singlet
 triplet or triplet
 singlet
Changes in spin multiplicity are forbidden http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/lumin1.htm

Selection rules
2.
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
3.
Selection rule of
D ℓ = ± 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.

s and s
* orbitals http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a

p and p
* orbitals http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a

Electronic Transitions: p  p
* http://www.cem.msu.edu/~reusch/VirtualText
/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 http://classes.kumc.edu/grants/dpc/instruct/index2.htm

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 “lowlying” p
* orbitals http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a

Electronic Transitions: n
 p
* http://www.cem.msu.edu/~reusch/VirtualText
/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 10 3 times smaller than e for the pp
* transition as it is “symmetry forbidden”.
McGarvey and Gaillard, Basic Photochemistry at http://classes.kumc.edu/grants/dpc/instruct/index2.htm

Lycopene from Tomatoes http:// www.purdue.edu/UNS/html4ever/020617.Handa.lycopene.html

• A plot of absorption versus wavelength is the absorption spectrum for two
A total l
 measurment
A
A so l
1 l
 
2


 l
1 l  l
-



 e e

A component e l
M
M l
1
M l
2 e
M l
1 l
N
2
 s
M under
  e
A
1 l
N
2
A l
N



 e e e e
 l
N l
1
N
2 l
N
1
2
M l
2 e

M
 
 
A e
M l
2 l
N
1

 l and
 




N

 e system l
N
wavelengt h l
 

 l e M l
 
 e l
N
 

 l
1 


 e M l
1 e M l
1
A
2 e l
N
2

 e M l
2 e M l
2
A
1 e l
N
1





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 0.717 at 240 nm and 0.239 at 280 nm.
Estimate the content of tryptophan and tyrosine in mol (g protein) -1



5380
11300

0 .
717

5380
 
 
1960
1500

0 .
239

1960



5 .
85

10

5
M



11300
11300


0 .
239
5380
 
 
1500
1500


0 .
717
1960



2 .
81

10

5
M

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.

e M l
 e l
N  e iso l 
iso

 e iso l
 
 e iso l
  e iso l
     

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

1.
Change in local charge distribution
2.
Change in dielectric constant
3.
Change in bonding interaction
4.
Change in dynamic coupling between different parts of the molecule

http://www2.mrc-lmb.cam.ac.uk/groups/GS/eye.html
Retina
Light sensitive protein Outer segment
Retina

1BRD http://www2.mrc-lmb.cam.ac.uk/groups/GS/rmovie.html
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 alltrans 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
1BM1