Absorption Spectroscopy

By
Srinivas Acharya
Lecturer, Env. Science
Visible & near-UV region wavelength (nm)
Microwave & radiowave region frequency (Hz)
Infared region wavenumber (cm-1)
Far-UV, x-ray, -ray energy (=h)
 Absorption & Emission

Rapid process(10-15s)
Absorption & Emission
 Radiation-Induced Transition
c
I      
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 () 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: I0 = intensity of incident light
I = intensity of transmitted light
 = molar absoptivity coefficient in cm2
log(I0/I) = bc mol-1
c = concentration in mol L-1
 = A/cb
b = pathlength of absorbing
A = bc solution in cm-1
A = absorbance = log(Io/I)
A = c (when b is 1 cm)

0.1
cm
 Beer-Lambert Law
I0
A  log  bl   log T
It
• A Absorbance or optical density (OD)
• absorptivity; M-1 cm-1
• c concentration; M
• T transmittance
Transmittance, Absorbance, and Cell
Pathlength
 Deviations from the Beer-Lambert Law

Low
c

High
The Beer-Lambert law assumes that
c
all molecules contribute to the
absorption and that no absorbing
molecule is in the shadow of another
 Sample Concentrations

Solution too concentrated Diluted five-fold

 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 (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
 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
 UV-visible spectroscopy definitions

chromophore Any group of atoms or part of a molecule 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.

How to impress your friends and family!
Making Molecular Orbitals
Antibonding

Bonding

In this case, the energies of the A.O.’s are identical

Absorption and Emission of Photons
 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.
Singlet and Triplet Excited States
Absorption and emission pathways

McGarvey and Gaillard, Basic Photochemistry

 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
 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
and * orbitals
 Electronic Transitions:   *

The   * transition involves orbitals that

have significant overlap, and the probability is
near 1.0 as they are “symmetry allowed”.

McGarvey and Gaillard, Basic Photochemistry

 * 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
 Electronic Transitions: n  *

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”.
McGarvey and Gaillard, Basic Photochemistry
 Molecular Orbitals in H2
The next-lowest energy orbital is unoccupied. As it lies above the highest
atomic orbital, we refer to it as an anti-bonding orbital.

R= 0.735 Look also at the shape of the lobes:

The anti-bonding orbital has a node between the
(H) Å two nuclei.
(H2)
Where the bonding orbital has an electron density
2s
build-up between the nuclei, the anti-bonding
orbital would have a reduced
electron density (2).
This orbital is called the Lowest
Unoccupied Molecular Orbital
 (LUMO)

1s 
This orbital is called the Highest
  Occupied Molecular Orbital
(HOMO)

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
 
Atotal  AM  AN   M l M   N l N   l  M M   N N 
measurments under 2 wavelength
 
A 1  l  M1 M   N1 N 
 
A 2  l  M2 M   N2 N 
so
1  2 1 1 A2 
 N N
 A  
M    M N M N 
l     
 1 2 2 1 

  M
1  1 2A   M
2 A1

N   M N M N 

l     
 1 2 2 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
•Molar absorptivity Ɛtry=11300 mol-1 cm2 and Ɛtyr=5380 mol-1 cm2

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

tyr   5380  0.717  1960  0.239

 5.85  10 5 M
11300  5380  1500 1960
tyr   11300  0.239  1500  0.717
 2.81 10 5 M
11300  5380  1500 1960
 Isosbestic points
Isosbestic wavelength is 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
M N
       iso
  isosbestic : A iso   iso l M   iso l N 
  iso l M  N 

In spectroscopy, an isosbestic point is a specific

wavelength, wavenumber or frequency at which the total
absorbance of a sample does not change during a chemical
reaction or a physical change of the sample.
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
11
Human Eye
http://www2.mrc-lmb.cam.ac.uk/groups/GS/eye.html

Retina

Light sensitive
Retina
protein Outer segment
 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
1BRD impulse which is
transmitted to the brain
http://www2.mrc-lmb.cam.ac.uk/groups/GS/rmovie.html
by the optical nerve
1BRD
1BM1