absorption spectroscopy of biopolymers overview
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Visible & near-UV region wavelength (nm)
Microwave & radiowave region frequency (Hz)
Infared region wavenumber (cm-1)
Far-UV, x-ray, -ray energy (=h)
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 () between 190 and 800 nm
UV-visible spectrum
The two main properties of an absorbance peak are:
1. Absorption wavelength
max
2. Absorption intensity
Amax
Housecroft and Sharpe, p. 466
Beer-Lambert Law
Beer-Lambert Law:
log(I0/I) = bc
= A/cb
A = bc
A = c (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)
0.1 cm
ℓ
http://www.hellma-worldwide.de/en/default.asp
Beer-Lambert Law
• A Absorbance or optical density (OD)• absorptivity; M-1 cm-1
• c concentration; M • T transmittance
TblI
IA
t
loglog 0
Transmittance, Absorbance, and Cell Pathlength
http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm
Deviations from the Beer-Lambert Law
The Beer-Lambert law assumes that all molecules contribute to the absorption and that no absorbing molecule is in the shadow of another
Low c
High c
http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm
Molar absorptivities ()
Molar absoptivities are very large for strongly absorbing chromophores ( >10,000) and very small if the absorption is weak (= 10 to 100). The magnitude of 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
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv2
UV-visible spectrum of 4-nitroanaline
Solvent: Ethanol
Concentration: 15.4 mg L-1
Pathlength: 1 cm
NH2
NO2
Molecular mass = 138
Harwood and Claridge, p. 18
UV-visible spectrum of 4-nitroanaline
1. Determine the absorption maxima (max) and absorption intensities (A) from the spectrum:
max = 227 nm, A227 = 1.55 max = 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
3. Determine the molar absorptivity coefficients () from the Beer-Lambert Law: = A/cℓ
227 = 1.55/(1.0 cm x 1.12 x 10-4 mol L-1) = 13,900 mol-1 L cm-1
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
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.
Absorption
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: 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 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.
and* orbitals
http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a
Electronic Transitions: *
McGarvey and Gaillard, Basic Photochemistry at http://classes.kumc.edu/grants/dpc/instruct/index2.htm
The * transition involves orbitals that have significant overlap, and the probability is near 1.0 as they are “symmetry allowed”.
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv2
* 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” * orbitals
http://www.cem.msu.edu/~reusch/VirtualText/intro3.htm#strc8a
Electronic Transitions: n *
McGarvey and Gaillard, Basic Photochemistry at http://classes.kumc.edu/grants/dpc/instruct/index2.htm
The n-orbitals do not overlap at all well with the * orbital, so the probability of this excitation is small. The of the n* transition is about 103 times smaller than for the * transition as it is “symmetry forbidden”.
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/uvspec.htm#uv2
Quantitative Analysis• A plot of absorption versus wavelength is the absorption spectrum
NMNM
MM
NMNM
NN
NM
NM
NMNMNMtotal
AA
l
AA
l
NMl
NMl
NMlNlMlAAA
1221
21
1221
12
222
111
12
21
1N
1M
soA
Ah wavelengt2under smeasurment
systemN and Mcomponent -for two
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
Mtyr
Mtyr
5
5
1081.219601500538011300
717.01500239.011300
1085.519601500538011300
239.01960717.05380
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.
Origin of Spectroscopic Changes
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
Human Eye
RetinaRetina
RetinaRetina
Outer segmentOuter segmentLight sensitive Light sensitive
proteinprotein
http://www2.mrc-lmb.cam.ac.uk/groups/GS/eye.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 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 http://www2.mrc-lmb.cam.ac.uk/groups/GS/rmovie.html
1BRD