An electromagnetic wave

Electromagnetic spectrum
Absorption Spectroscopy

Energy level differences between

electronic levels: 102 kJ/mol
vibrational levels: 100 kJ/mol
rotational levels: 10-2 kJ/mol
 UV/Visible (Electronic) Spectroscopy

If light with suitable energy n hits a molecule in the ground state 1, the
energy can be absorbed and the molecule is raised to an electronically
excited state 2.

The molecule can return to its ground state via spontaneous emission of
light (a photon).

E = h

The valence electrons are the only ones whose energies permit them to
be excited by near UV/visible radiation.
Singlet and triplet excited states
 The origin of the absorptions

The energy of these electronic transitions is generally in the following order

 The origin of the absorptions

to * Transition: An electron in a bonding s orbital is excited to the

corresponding antibonding orbital. The energy required is large. (Methane:
125 nm)

n to * Transition: Saturated compounds containing atoms with lone pairs

(nonbonding electrons) are capable of n to * transitions. 150 250 nm.
 The origin of the absorptions

n to * and to * Transitions:

Most absorption spectroscopy of organic compounds is based on

transitions of n or electrons to the * excited state. This is because the
absorption peaks for these transitions fall in an experimentally convenient
region of the spectrum (200 - 700 nm). These transitions need an
unsaturated group in the molecule to provide the electrons.
 The origin of the absorptions

*
High energy required, vacuum UV range
CH4: = 125 nm
n *
Saturated compounds, CH3OH etc ( = 150 - 250 nm)
n * and *
Mostly used! = 200 - 700 nm

Chromophore:

In a number of molecules, the absorption of a photon can be traced to the

excitation of electrons of a small group of atoms.
Example: C=O group, absorption around 290 nm.
 The origin of the absorptions

Biological chromophores

The peptide bonds and amino acids in proteins

o The electrons of the peptide group are delocalized over

the carbon, nitrogen, and oxygen atoms. The n-* transition
is typically observed at 210-220 nm, while the main -*
transition occurs at 190 nm.

o Aromatic side chains contribute to absorption at > 230 nm

Purine and pyrimidine bases in nucleic acids and their derivatives

Highly conjugated double bond systems

 Interactions between chromophores

A molecule which contains several isolated chromophores will give a UV-vis

spectrum which essentially consists of the additive absorptions of the
individual groups.

This is not the case for conjugated chromophores: With increasing

conjugation the absorption energy and intensity decreases steadily
A UV/Visible absorption experiment
UV/Visible absorption spectrophotometers

A single-beam spectrophotometer

Requires a stabilized voltage supply

UV/Visible absorption spectrophotometers

A double-beam (space-resolved) spectrophotometer

Need two detectors

UV/Visible absorption spectrophotometers

A double-beam (time-resolved) spectrophotometer

 Light sources for UV/Vis spectroscopy
For Visible and IR
- a tungsten lamp
- operates at 3000 K
- produces l from 320 to 2500 nm

For UV:
- a common lamp is a deuterium arc lamp
- electric discharge causes D2 to dissociate and emit UV radiation (160 325 nm)
- other good sources are:
Xe (250 1000 nm) Hg (280 1400 nm)
 Beer-Lambert Law

dl

Molar absorption
Absorbance Coefficient
Depends on wavelength
 Molar absorption coefficient and cross sections

Assumption: Molecule is an opaque disc of cross-section area,

Taking an infinitesimal slab, d of the sample

Then, the total opaque area on the slab due to the absorbers = N A d

The fraction of photons absorbed, dI /I = ( N A d ) / A

 Molar absorption coefficient and cross sections

Therefore, for a chromophore with cross sectional area 12, the maximal value (assuming
that the transition probability is 1) is 2.6 x 104 M-1cm-1.
 Deviations from Beer-Lambert law

Low c

High c

Solution too concentrated Diluted five-fold

Deviations from Beer-Lambert law

Polychromatic light
 Absorption Spectroscopy

Absorption: a femtosecond phenomenon

Franck-Condon principle
 Biological chromophores

The peptide bonds and amino acids in proteins

The electrons of the peptide group are delocalized
over the carbon, nitrogen, and oxygen atoms. The n-
* transition is typically observed at 210-220 nm,
while the main - * transition occurs at 190 nm.
Aromatic side chains contribute to absorption at >
230 nm

Purine and pyrimidine bases in nucleic acids and

their derivatives

Highly conjugated double bond systems

 Biological chromophores

Amino acids

Molecule (nm) (M-1cm-1)

Tryptophan 280, 219 5600, 47000

Tyrosine 274, 222, 193 1400, 8000, 48000

Phenylalanine 257, 206, 188 200, 9300, 60000

Histidine 211 5900

Cystine 250 300

 Biological chromophores

Bases and their derivatives

Molecule (nm) (M-1cm-1)

Adenine 260.5 13400

Adenosine 259.5 14900

NADH 340,259 6230, 14400

NAD+ 260 5900, 18000

FAD+ 450 11300

 Biological chromophores

Highly conjugated double bond systems

Major absorption peaks around

400 and 540-580 nm are due to
* transitions of the
porphyrin ring
 Biological chromophores

Highly conjugated double bond systems

160000

120000

80000 Series1
-carotene
Absorption due to *
40000 transitions

0
380 480 580 680
 Biological chromophores

Highly conjugated double bond systems

 Rule of thumb for conjugation

If double bonds are greater than one single bond apart

- are relatively additive (hyperchromic shift)

- constant

CH3CH2CH2CH=CH2 max= 184 max = 10,000

CH2=CHCH2CH2CH=CH2 max=185 max = 20,000

If conjugated

- shifts to higher s (red shift)

H2C=CHCH=CH2 max=217 max = 21,000

 Effects of solvents

Blue shift (n *) (Hypsocromic shift)

o Increasing polarity of solvent better solvation of electron pairs (n

level has lower E)

o peak shifts to the blue (more energetic)

o 30 nm (hydrogen bond energy)

Red shift (n * and *) (Bathochromic shift)

o Increasing polarity of solvent, then increase the attractive polarization

forces between solvent and absorbing species, thus decreases the
energy of the unexcited and excited states, with the later greater.

o peaks shift to the red

o 5 nm
Spectral nomenclature of shifts
 Hyperchromic effect in unstacked bases

Hyperchromic effect Melting of DNA

Multichannel design for HPLC

2
 Sample purity

Pure DNA, A260/280 ~1.8 Pure RNA, A260/280 ~2

260:280 ratio has high sensitivity for nucleic acid contamination in protein
% protein % nucleic acid 260:280 ratio
100 0 0.57
95 5 1.06
90 10 1.32
70 30 1.73

260:280 ratio lacks sensitivity for protein contamination in nucleic acids

% nucleic acid % protein 260:280 ratio
100 0 2.00
95 5 1.99
90 10 1.98
70 30 1.94
 Fluorescence Spectroscopy

The processes that occur between the absorption and emission

of light are usually illustrated by the Jablonski diagram.

Vibrational
relaxation

Jablonski diagram

Typical lifetimes of electronic processes

Absorption: 10-15 s Internal conversion and vibrational relaxation: 10-12 s

Fluorescence: 10-9 10-8 s Phosphorescence: 10-3 -100 s
 Molar absorption
coefficient (M-1 cm-1)
Fluorescence Spectroscopy

Fluorescence Intensity (Normalized)

 Fluorescence Spectroscopy

Fluorescence typically occurs from aromatic molecules

 Characteristics of fluorescence emission

Stokes shift

Kashas rule

Mirror-image rule
 Characteristics of fluorescence emission

Stokes shift: energy of the emission is typically less than that of

absorption.

Internal conversion and vibrational relaxation

Emission to higher vibrational levels

Solvent reorientation
 Characteristics of fluorescence emission
Solvent reorientation

o Rotational time scale of small solvent molecules: 40 ps or less.

o Ample time for the solvent molecules to reorient around the excited-
state dipole, which lowers its energy.

o The process is called solvent relaxation and occurs in 10-10 s in fluids.

 Characteristics of fluorescence emission

Kashas rule: Emission spectra are typically independent of the excitation

wavelength.

Internal conversion (fluorescence is from S1)

o Exceptions exist and some molecules

emit from S2
o Emission from S2 not observed in
biomolecules

Vibrational relaxation
Mirror image rule and Franck-Condon principle

Mirror image rule: The emission is the

mirror image of the S0 S1 absorption.

Similarities of the vibrational levels of S0

and S1: vibrational energy levels are not
significantly altered by the different
electronic distributions of S0 and S1.

Franck-Condon principle: All electronic

transitions are vertical.

If a particular transition probability

(Franck-Condon factor) between the 0th
and 1st vibrational levels is largest in
absorption, the reciprocal transition is
also most probable in emission.
Mirror image rule

Why perylene follows the mirror

image rule, but quinine emission lacks
the two peaks seen in its excitation
spectrum at 315 and 340 nm?

Because, in the case of quinine, the

shorter wavelength absorption peak
is due to excitation to the second
excited state (S2), which relaxes
rapidly to S1.

Therefore, the emission is the mirror

image of S0 S1, absorption, not of
the total absorption spectrum.
 Exceptions to the Mirror image rule

pH sensitive fluorophore

Formation of excited-state complexes, 1-hydroxypyrene trisulfonate

called exciplexes

Formtion of excited-state dimer called

excimer.

Other excited state reactions

Anthracene
Fluorescence emission spectra of aromatic amino acids
 Fluorescence lifetimes and quantum yields

Rate of fluorescence

Rate of non-radiative decay

A simplified Jablonski diagram

Fluorescence quantum yield: Ratio of number

of photos emitted to the number absorbed

Fluorescence lifetime: The average time spent

by a fluorophore in the excited state
 Fluorescence lifetimes
Suppose a sample containing the fluorophore is excited with an
infinitely sharp (-function) pulse of light.

This results in an initial population (n0) of fluorophores in the excited

state.

Emission is a random event, and each excited fluorophore has the

same probability of emitting in a given period of time.

In a fluorescence experiment we do not observe the number of

excited molecules, but rather fluorescence intensity, which is
proportional to n(t)
 Meaning of the Lifetime
The lifetime is the average amount of time a fluorophore remains in
the excited state following excitation.

This can be seen by calculating the average time in the excited state
<t>.

<t> is obtained by averaging t over the intensity decay of the

fluorophore

The denominator is . Integration by parts shows that numerator is 2.

True for single exponential decay

Lifetime is a statistical average, and fluorophores emit randomly

throughout the decay.
 Fluorescence quenching
Fluorescence quenching refers to any process that decreases the fluorescence
intensity of a sample.

Molecular interactions that can result in quenching: excited-state reactions,

molecular rearrangements, energy transfer, ground-state complex formation,
and collisional quenching.

Static quenching: In static quenching a complex is formed between the

fluorophore and the quencher, and this complex is nonfluorescent.

Dynamic/collisional quenching: occurs when the excited-state fluorophore is

deactivated upon contact with some other molecule in solution, which is
called the quencher.
 Collisional quenching

F0/F is given by the ratio of the decay rate in the absence of quencher
() to the total decay rate in the presence of quencher ( + kq[Q]):

Since collisional quenching is a rate process that depopulates the

excited state, the lifetimes in the absence (0) and presence () of
quencher are given by

Therefore 2

From equations 1 and 2,

Stern-Volmer equation
Fluorescence quenching
 Fluorescence lifetime
Fluorescence lifetime, is the time it takes for intensity to decay to I0/e

Distinguishing between static and dynamic quenching using lifetime measurements

Static quenching: Fluorescence lifetime is not changed

Dynamic/collisional quenching: Fluorescence lifetime

decreases with increase in quencher concentration
 Fluorescence lifetime
Biopolymers display multi-exponential or heterogeneous decays