Absorption Spectroscopy of
Biopolymers
Overview
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)
Absorption & Emission
Rapid process(10-15s)
Absorption & Emission
Radiation-Induced Transition
c
I n    n 
n
• Absorption
• Stimulated emission
• Spontaneous emission
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
UV-visible spectrum
The two main properties of an
absorbance peak are:
1. Absorption wavelength
lmax
2. Absorption intensity
Amax
Housecroft and Sharpe, p. 466
Beer-Lambert Law
Beer-Lambert Law:
I0 = intensity of incident light
I = intensity of transmitted light
log(I0/I) = ebc
e = molar absoptivity coefficient in cm2
mol-1
e = A/cb
c = concentration in mol L-1
b = pathlength of absorbing
A = ebc
solution in cm-1
A = ec (when b is 1 cm)
A = absorbance = log(Io/I)
ℓ
0.1
cm
http://www.hellma-worldwide.de/en/default.asp
Beer-Lambert Law
I0
A  log  ebl   log T
It
•
•
•
•
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
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
http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/beers1.htm
Sample Concentrations
Solution too concentrated
Diluted five-fold
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 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
NH2
NO2
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 (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
3. Determine the molar absorptivity coefficients (e) from the BeerLambert 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
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: DS = 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 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.
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 103
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
Chlorophyll
B-carotene
hemoglobin
Quantitative Analysis
• A plot of absorption versus wavelength is the absorption spectrum
for two - component M and N system
Altotal  AlM  AlN  e lM l M   e lN l N   l e lM M   e lN N 
measurment s under 2 wavelength
A l1  l e lM1 M   e lN1 N 
A l2  l e lM2 M   e lN2 N 
so
N
N

e
A

e
1  l2 1 l1 A2 
M    M N M N 
l el el  el el
2
1 
 1 2


M
M

e
A

e
1  l1 2
l2 A1
N    M N M N
l el el  el el
2
1
 1 2








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  0.717   1960  0.239 
tyr 
 5.85 10 5 M
11300  5380   1500 1960 

11300  0.239   1500  0.717 
tyr 
 2.81 10 5 M
11300  5380   1500 1960 
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.
Isosbestic points
e l  e l  e iso
M
N
l  isosbestic: A iso  e isol M   e isol N 
 e isol M   N 
UV spectrum of BSA
UV spectrum of DNA
from E. coli
UV Absorption of amino acid
Effect of Secondary structure
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
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