MOLECULAR SPECTROSCOPY 01

EXPERIENCE THROUGH MULTIPLE EXCITATIONS

MOLECULAR SPECTROSCOPY RECALLING THE LIGHT!

Molecular spectroscopy deals with the interaction of the light with molecules resulting in
various types of excitations and spectra. As shown below, there are various techniques
depending on the type of interacting energy:

UV

Far UV

10

IR

Near UV

180

Visible

350
nm

Near IR

770

Mid IR

2500
2.5

Far IR

50
m

Microwave

1000
1

Radio wave

300
mm

>300

MOLECULAR SPECTROSCOPY UV/VISIBLE SPECTRA

Theory: The electronic absorption
In atomic spectroscopy the absorption of energy leads to excitation of valence shell
electrons to higher energy levels of free atoms or ions. The observed spectra is not
complicated, the outcome of which being simpler line spectra.
In electronic spectra the absorption of energy leads to excitation of bonded electrons
(in some cases non-bonded electrons) within the molecule. The radiation which causes the
electronic
l t i excitation
it ti mustt b
be iin th
the hi
higher
h energy region
i off th
the electro-magnetic
l t
ti spectrum,
t
ie.
i
UV and Visible region. Hence the technique is called as UV/Visible Spectroscopy. There are
three distinct types of electronic transitions in organic molecules as follow:
1. The electrons involved in saturated bonds (covalent bonds), such as those found in C-C,
C-H, etc. These bonds are called as sigma () bonds.
2. The electrons involved in unsaturated bonds, such as those found in C=C, C=O, etc and are
called as pi () bonds.
3 The electrons that are not involved in bond formation (nonbonding or unshared) and are
3.
localized within such atoms as oxygen, nitrogen, sulfur and halogens. These electrons are
termed as n electrons.

MOLECULAR SPECTROSCOPY UV/VISIBLE SPECTRA

Transitions: In -bond formation, individual atoms will share the electrons through their
hybridized atomic orbitals by direct overlapping to form a low-energy
low energy bonding molecular
orbital () and a high-energy anti-bonding orbital (*).
*

In -bond formation, the atomic orbitals will combine through parallel overlap to form a low
energy bonding molecular orbital () and a high-energy anti-bonding orbital (*).
*

In addition to and bonds many organic compounds contain non-bonding

non bonding electrons (n),
(n)
which do not participate in bond formation but can be excited to higher energy levels.
n

n
n

*
*
*
*

MOLECULAR SPECTROSCOPY UV/VISIBLE SPECTRA

*
*
E

The energy scale for these transitions are as:

n

* > n

* >

* > n

* Transitions: The energy required to induce this transition is higher compared to other
transitions. Thus, saturated hydrocarbons such as methane and ethane absorb at 125nm and 135
nm respectively.
p
y Normally
y these transitions occur below 200nm.
n
* Transitions: Saturated compounds containing unshared electron pairs are capable of
these transitions. These transitions require less energy than the previous one and occur in the
region
g
160-260nm.

* and n
* Transitions: Most applications of UV/Visible absorption spectroscopy arise
from these two types of transitions which fall in the region 200 700nm This range is an
g
experimentallyy convenient region.
These transitions require the presence of an unsaturated pair of electrons and functional groups
(double and triple bonds) to provide the orbitals.

UV/VISIBLE SPECTRA CHROMOPHORES/AUXOPHORES

The unsaturated absorbing centers are termed as chromophores. (Though the term
chromophore is strictly used for unsaturated centers, this can be also used to describe the
groups in general responsible for the absorption). Examples are: C=C, C=O and N=N
There are another set of groups called as auxophores,
auxophores which themselves dont
don t absorb,
absorb but
when conjugated to chromophores causes a shift in the absorption wavelength. Examples are:
OH, Br, and NH2. The following table shows chromophores and auxophores, and their
corresponding
p
g absorptions
p
in wavelength.
g
Thus the absorption characteristics of a molecule can often be predicted from the presence of
chromophores in a molecule. Similarly, the absorption spectrum of an unknown can be used to
identify the presence of certain functional groups in a molecule.
For example, the absence of absorption bands in the region 200-800nm indicates the absence
of chromophores (and unsaturated bonds) and thus it can be predicted that the compound
under study could be a saturated organic compound or an inorganic compound with no
double bonds.
The wavelength at which the absorption is maximum is termed as max

UV/VISIBLE SPECTRA - CHROMOPHORES AND ABSORPTIONS

Chromophore

Example

Type of Transition

max

Alcohol

CH3-OH

167nm

Alkene

-C=C-, CH2=CH2

170nm

Alkyne
y

-CC-,, CHC-CH2-CH3

172nm

Amido

CH3

214nm

C=O
NH2
Amine

CH3-NH2

215nm

Azo

CH3-N=N-CH3

339nm

Carbonyl

CH3

280nm

204nm

C=O
CH3
Carboxyl

CH3
C=O
C
O
OH

Nitrate

C2H5-ONO2

270nm

Nitro

CH3-NO2

280nm

UV/VISIBLE SPECTRA - CHROMOPHORES AND ABSORPTIONS

Study
y of simple
p organic
g
molecules

CH3-Cl : 172nm, CH3-Br : 204nm, CH3-I : 258nm (Transition type: n

(larger the size of the halogen atom more easier is the transition)

(CH3)2 CO: 280nm, (CH3)2 CS: 400nm (Transition type: n

(oxygen is more electronegative than sulfur)

*)

*)

(CH3)2CO: 280nm, CH3CONH2: 214nm, CH3COOH: 204nm (Transition type: n

(lone pair of electrons from N and O interacts to increase the * energy state)

CH2=CH2: 170nm, CH2=CHNH2: 220nm (Transition types:

( h
(change
iin ttransition
iti ttype))

* & n

*)

C2 H5
CH2

CH2

=170nm;

CH3

(CH2 ) 5

CH = CH2

*)

C=C

=177nm;
H

=184nm
C2H5

UV/VISIBLE SPECTRA - CHROMOPHORES AND ABSORPTIONS

Study
y of aromatic system
y
Benzene ring has three absorption peaks due to
* at: 184, 204 and 256nm. All three peaks
are strongly affected by substituents which act as auxophores.
The substituent effects in benzene ring are mainly due to resonance interactions and inductive
effects. Some of the auxophores in benzene ring and their absorptions are given below
(in comparison with 256nm):
-NH2: 280nm, -OH: 270nm, -Cl: 265nm, -COOH: 273nm, CN: 271nm, -CH=CH2: 282nm
-COCH3: 278nm (320nm due to n

*)

??

UV/VISIBLE SPECTRA - CHROMOPHORES AND ABSORPTIONS

Study
y of conjugated
j g
systems:
y
-(C=C)
(
)2- : 210-230nm; -(C=C)
(
)3- : 260nm; -(C=C)
(
)5- : 330nm

E1

E2

E3

E1 > E2 > E3

UV/VISIBLE SPECTRA - CHROMOPHORES AND ABSORPTIONS

Effect of conjugation
j g
More the number of conjugated double bonds in a molecule, the longer will be the wavelength of
maximum absorption.
CH2=CH2: 170nm, CH2=CH-CH=CH2: 217nm, CH2=CH-CH=CH-CH=CH2: 258nm
CH3N=NCH3: 345nm, C6H5N=NC6H5: 445nm, C6H5CH2N=NCH2C6H5: 295nm
As the number of -CH=CH- group increases the absorption shifts to longer wavelengths.
Introduction of a CH2 group in the chain disturbs the conjugation and shifts the wavelength to
shorter region
region.
However substitutions (like that of alkyl groups) has no appreciable impact on the absorption:
CH3-CH=CH-CH=CH
CH CH CH CH2: 224nm

CH3-CH=CH-CH=CH-CH
CH CH CH CH CH3: 227nm

CH2=CH-C(CH3)=CH2: 224nm

CH2=C(CH3)-C(CH3)=CH2: 226nm

UV/VISIBLE SPECTRA - CHROMOPHORES AND ABSORPTIONS

Highly
g y conjugated
j g
systems
y
are colored

Isoprene is colorless

max = 222nm

Isoprene has a max of 222nm and is colorless but as the

number of isoprene units increases the absorption shifts
to longer wavelengths and color is observed.

max = 463nm
463
-Carotene

max = 476 nm
Lycopene

UV/VISIBLE SPECTRA - CHROMOPHORES AND ABSORPTIONS

Other systems
y

= 182nm

= 256nm

= 185nm ;

= 260nm;

= 295nm ;

C=C

= 280nm

C=C
C6H5

C6H5

O
CH3

= 280nm;

= 300nm

= 380nm;

C6H5

= 273nm;

= 314nm;

C6H5

CH3

= 235nm;

CH3

= 320nm;

= 348nm

UV/VISIBLE SPECTRA REPRESENTATIVE SPECTRUM

max

UV/VISIBLE SPECTRA RECALLING THE BEER-LAMBERT LAW

A bc or A = abc,
where A = absorbance
where,
a = absorption coefficient
b = optical path length
c = concentration of substance in the test solution

For multiple species:

Atotal = xcxb + YcYb +

When c is expressed in mol.L-1 and b in cm, then a is called the molar absorptivity or molar
absorption coefficient. Thus the above equation may be re-written as, A = bc, has the unit:
L.mol-1.cm-1. For strongly absorbing molecules in UV/Vis region, can be as high 10,000 or
even 100,000. The value of molar absorptivity is an indicator of the presence of particular functional
group.
Deviation in Beers law:
At high
g concentration the average
g distance between the molecules is reduced and each molecule
affects the charge distribution of its neighbors, which in turn affects the absorbing ability of the
molecules at a given wavelength.
The molar absorption coefficient is dependent on the refractive index of the medium. Thus, if
concentration changes cause significant alterations in the refractive index of a solution, deviation
will occur.

UV/VISIBLE SPECTRA SOLVENT CUT-OFFS

Solvent
Water
Acetonitrile
n-Hexane
isopropanol
Methanol
Ethanol
Cyclohexane
Diethyl ether
1,4-Dioxane
Tetrahydrofuran
Chloroform
Benzene
T l
Toluene
Acetone

Cut-Off
Wavelength (nm)
190
190
195
205
206
210
210
220
220
230
245
280
285
330

Water is
W
i a good
d solvent
l
for
f
UV/Vis spectroscopy

Sampling Cells:
Quartz for UV range
Glass for visible range
For the entire UV-Visible range:
Quartz is the choice

UV/VISIBLE SPECTRA SOLVENT EFFECTS

Certain compounds tend to interact with selected solvents with a change in the absorption
wave lengths.
g
Bathochromic shift mostly observed in to * transitions and corresponds to shift to longer
wavelength. It is also termed as red shift.
Attractive polarization forces between the solvent (polar solvent) and the compound, through
dipole-dipole interactions, tend to lower the energy levels of both the ground and excited states.
This results in the reduction of energy
gy difference between them leading
g to absorption
p
in longer
g
wavelength (lower energy). This effect is more associated with those compounds containing
unsaturated bonds (-C=C-).

UV/VISIBLE SPECTRA SOLVENT EFFECTS

Hypsochromic
yp
shift mostlyy observed in n to * transitions and corresponds
p
to shift to
shorter wavelength. It is also termed as blue shift.
This arises from the solvation of the non-bonded electron pair, through H-bonding, which lowers
ONLY the energy
gy level of the n orbital. This increases the energy
gy g
gap
p between the g
ground and
excited states with subsequent absorption in shorter wavelength (higher energy). This effect is
more in compounds with non-bonding electrons (-C=O-).

Note: If a molecule contains both transitions, then it will exhibit both effects correspondingly.

UV/VISIBLE SPECTRA SOLVENT EFFECTS

Hypsochromic shift

Bathochromic shift
Hypochrom
mic effect

Intensity
y

Hyperchrromic effect

There are two other terms as hyperchromic for greater absorbance and hypochromic for
lower absorbance, which are due to the structural changes of the analyte molecule. These two
effects occur at the same wavelength.

Wavelength

UV/VISIBLE SPECTRA OTHER TRANSITIONS

UV/visible
UV/
i ibl spectra are also
l possible
ibl due
d to d and
d f electron
l
transitions
ii
and
d through
h
h charge
h
transfer
f
absorptions:
d and f electron transitions:
F d electrons
For
l t
mostly
tl 3d and
d 4d electrons
l t
are responsible
ibl from
f
th first
the
fi t and
d second
d transitiont
iti
+2
+2
+2
+3
+3
metal series such as, Fe , Cu , Mn , Cr , V , etc.
The individual ions and their complexes tend to absorb leading often to a broad absorption bands
which
hi h are much
h affected
ff t d by
b chemical
h i l environmental
i
t l factors,
f t
such
h as the
th type
t
off ligands.
li
d
Thus Cu+2 in water as aquo complex is pale blue in color, while in ammonia it is much darker due to
copper-ammonia complex.
The spectral characteristics of transition metals involve electronic transitions among the various
energy levels within the d orbitals.
For f electrons
F
l t
mostly
tl 4f and
d 5f electrons
l t
are responsible
ibl from
f
th lanthanide
the
l th id and
d actinide
ti id series
i
such as Ho+3, Er+3, Pr+3, etc.
In distinct contrast to the behavior of most organic and inorganic absorbers, their spectra consist of
narrow, well-defined
ll d fi d and
d characteristic
h
t i ti absorption
b
ti peaks
k which
hi h are least
l
t affected
ff t d by
b the
th type
t
off
ligands associated.

UV/VISIBLE SPECTRA OTHER TRANSITIONS

Charge transfer absorptions:

Charge transfer absorptions mainly occur in inorganic complexes.
In such a complex one of its components will have electron-donor characteristic while the other
component electron-acceptor property.
Absorption of radiation then involves transfer of an electron from the donor to acceptor orbital. In
most charge-transfer complexes the metal serves as the electron acceptor.
An example is Fe+3-thiocyanate complex where the absorption of a photon results in the transfer of
an electron from the thiocyanate ion to an orbital associated with Fe+3 ion.

UV/VISIBLE SPECTRA INSTRUMENTATION AND APPLICATIONS

Instrumentation:
The instrument which is used to record the electronic spectra is called as UV/Vis
Spectrometer. It consists of a dual light source one for ultra-violet range (deuterium discharge
lamp) and another for visible range (tungsten lamp), monochromator, photodetector and optical
mirrors.
mirrors
For measurements cells (cuvettes) with a path length of 1cm is used. For visible range
normal g
glass cell can be used but for UV range
g aq
quartz cell must be used since at UV range
g
normal glass will have absorption due to inorganic ions.
A double beam spectrometer is used one beam path for the reference solvent and another for
the sample + solvent.
Reference cell
PC System

Detector

Source

Beam splitter

Sample cell

UV/VISIBLE SPECTRA INSTRUMENTATION AND APPLICATIONS

Applications:
- It is the most widely used technique for quantitative analysis
- To some extend in qualitative analysis through an absorption spectrum which is unique for a
compound.
- It is more often used in conjunction with other techniques in the identification and structural
analysis of newly synthesized organic compounds.
Disadvantages:
g
- Samples should be in solution
- Solutions should be transparent (ie. It should not be cloudy),
- mixture of compounds is difficult to analyze and requires prior separation.
- Require large sample volume
- Only one sample at a time

UV-Vis Spectrometers

- Reflective UV-Vis (suitable for reflective samples)

- Small-volume cuvettes (100 L), Nanodrop
- Microplate reader (for 96-well plate)

Microplate reader

Nanodrop

MOLECULAR SPECTROSCOPY LUMINESCENCE SPECTRA

Theory: In electronic spectra the absorption of light by molecules to cause excitation to higher
levels is studied, luminescence spectra is concerned with the dissipation of this extra energy
to return back to ground state. It is of two types:
Dissipation of Energy
Radiative
(Luminescence)

Non-radiative
(vibrational, rotational and translational)

The molecular luminescence spectroscopy is a delayed emission technique and available as

two types as photoluminescence and chemiluminescence depending on the type of
excitations. In photoluminescence, excitation is occurred by absorption of light with subsequent
delayed emissions and the phenomenon give rise to two types as fluorescence and
Phosphorescence. In chemiluminescence, excitation is occurred by chemical reactions.
Luminescence Spectra

Photoluminescence
Fluorescence

Phosphorescence

Chemiluminescence

PHOTOLUMINESCENCE SPECTRA SPIN MULTIPLICITY

Singlet and Triplet States:
A molecular electronic state (ground or excited) in which all electron spins are paired is called a
singlet state. When one of a pair of electrons is excited to a higher energy level, either the spin
of the promoted electron is still paired with the ground state electron and is said to be excited
singlet state or the spin may un-paired and is said to be excited triplet state. These states are
represented as follows:

ground singlet state

excited singlet state

Fluorescence is singlet-to-singlet transition (S1

occur.

excited triplet state

S0 + hv). It requires 10-10 -10-6 s to

Phosphorescence is triplet-to-singlet, which can be represented as: (T1

It requires 10-44 - 10 s or more,
more to occur.
occur

S0 + hv).

PHOTOLUMINESCENCE SPECTRA THE JABLONSKI DIAGRAM

Transitions: The various activation (excitation) and deactivation (relaxation) processes leading
to the photo luminescence spectra can be depicted through an energy-level diagram as shown
below known as Jablonski diagram:
a

c
a

S2
S1

Absorption

T1

Fluorescence

Phosphorescence

S0

a = vibrational relaxation; b = internal conversion; c = inter-system crossing

S0 = singlet ground state; S1 = singlet excited state 1; S2 = singlet excited state 2; T1 = triplet excited state 1

PHOTOLUMINESCENCE SPECTRA REPRESENTATIVE SPECTRUM

Green = Excitation and Red = Emission (Compound: Anthracene)

PHOTOLUMINESCENCE SPECTRA INSTRUMENTATION AND APPLICATIONS

Instrumentation: The instrument which is used to record the luminescence phenomenon is
called as Luminescence
Luminescence Spectrometer
Spectrometer. Akin to UV/Vis Spectrometer it consists of a dual light
source one for ultra-violet range (deuterium discharge lamp) and another for visible range
(tungsten lamp) for exciting the molecules. More widely a Xe lamp is used whose radiation
covers the whole UV and visible range.
Other components include two monochromators, fluorescence/phosphorescence
detectors, optical lenses and optical mirrors. For measurements quartz cells (cuvettes) with a
path length
p
g of 1cm is used. The luminescence exhibited byy the sample
p is measured at right
g
angles to the incident beam.
Source

Excitation
Filter
Detector 2

Detector 1
Emission
Filter

Emission
Filter

Sample cell

PHOTOLUMINESCENCE SPECTRA INSTRUMENTATION AND APPLICATIONS

Applications:
- Molecular fluorescence is an important tool in the chemical analysis, though its counterpart
atomic fluorescence is rather less studied.
- However it is limited to selected compounds like benzene and fused benzene ring systems,
systems and
other unsaturated compounds.
- Metals can also be analyzed by their ability to form complexes with the organic ligands.
- It finds uses in the analysis of foods for vitamin content, since vitamins like riboflavin, niacin,
etc., exhibit fluorescence.
Disadvantages:
), and the transitions
- Fluorescence is never observed in wavelength below 250nm (*
n or *
.
concerned confined to either *
- Thus the application is very limited, to those which have such transitions.
- In most of the cases it requires dyes to form complexes which can fluoresce, which may lead to
errors in
i quantitative
tit ti analysis.
l i
Advantages:
- Very high sensitivity (vs UV-Vis spectra)

PHOTOLUMINESCENCE SPECTRA INSTRUMENT LAY OUT