Spectroscopy

Spectroscopy is the study of the interaction between radiation and matter as a

function of wavelength λ. It also can refer to interactions with particle radiation
or to a response to an alternating field or varying frequency ν.
A plot of the response as a function of wavelength or more commonly frequency
is referred to as a spectrum.

Spectrometry is the measurement of these responses and an instrument which

performs such measurements is a spectrometer or spectrograph

Applications ::

 Physical chemistry
 Analytical chemistry
 Organic and inorganic chemistry
 Astronomy……..
 Spectroscopy
Classifications

Nature of Excitation Measurement Processes Common Types

measured
 Fluorescence
 X-ray
 Electromagnetic  Absorption  Flame
 Electron  Emission  Visible
 Mass  Scattering  Ultra-violet
 Dielectric  Infrared
 Raman
 NMR
 Photoemission
 Mössbauer
 Types of Spectroscopy
Modern techniques for structure determination of unknown compounds include:

• Ultraviolet (UV)-visible spectroscopy corresponds to excitations of outer

shell electron between the energy levels that correspond to the molecular
orbital of the systems.
• Infrared (IR) spectroscopy measures the bond vibration frequencies in a
molecule and is used to determine the functional group. What functional
groups are present in the compound.
• Mass spectrometry (MS) fragments the molecule and measures the
masses. What is the size and formula of the compound.
• Nuclear magnetic resonance (NMR) spectroscopy detects signals from
hydrogen atoms and can be used to distinguish isomers. What is the
carbon-hydrogen framework of the compound.
• Fluorescence is the emission of photons from electronically excited states of
atoms, molecules, and ions it is used to determine the excited state property
of the molecule.
• MÖssbauer spectroscopy (or γ -ray spectroscopy) is concerned with
transitions between energy levels within the nuclei of atom. It can be used to
determine the valance state of metal in unknown compound.
• Raman effect may be defined as phenomenon due to which scattering of
light has a slightly different frequency from that of incident light and there
occurs a change in atomic oscillation within the molecule. It is helps in the
elucidation of molecular structure, for locating functional groups or chemical
bonds in the molecules.
 Spectroscopy and the Electromagnetic Spectrum

The electromagnetic spectrum covers a continuous range of

wavelengths and frequencies, from radio waves at the low-
frequency end to gamma (γ ) rays at the high-frequency end
higher frequency lower frequency
shorter wavelength longer wavelength
Spectroscopy and the
Electromagnetic Spectrum
Electromagnetic energy is transmitted in discrete amounts called
quanta
Amount of energy, , corresponding to 1 quantum of energy (1
photon) of frequency (ν )∈
is expressed by the Planck equation

hc
∈ = hν =
λ
The energy of a photon varies directly with its frequency but inversely with its
wavelength
-34
h = Planck's constant = 6.62 × 10 J ⋅s
E is energy of Avogadro's number ( N A )
of quanta ∈ , or, energy of one mole of photons
of wavelength λ

N A hc 1.20 × 10-4 kJ/mol

E= =
λ λ (m)

It is known as one Einstein of energy

 UV-Visible Spectroscopy

• Region of greatest interest to organic chemists from 2 x 10-7 m to ~8 x 10-7 meters

(i) Far (or vacuum) ultraviolet: The region from 10-200 nm can be studied in evacuated
systems and is termed as “vacuum ultraviolet”. The atmospheric absorption below
200 nm is a blessing to all, including the spectroscopists, since it prevents the
hazardous (high energy) ultraviolet radiation in the sunlight from striking the earth’s
surface.
(ii) Near or quartz ultraviolet : The region from 200-380 nm
The atmosphere is transparent in this region and quartz optics may be used to scan
from 200-380 nm.
(iii) Visible region: The region from 380-780 nm
A tungsten filament lamp is generally used for the visible region of the spectrum.
In a typical experiment, the molecules or atoms
start at lower energy and go to a higher energy
level upon absorption of radiation of appropriate
wavelength
After

n
Before it o
rp ∆ E
Energy

s o
Ab
Absorption can only occur when the energy of
the radiation (calculated from the frequency or
wavelength) matches the energy gap.

If there are several different upper levels (and

there usually are) then several transitions will be
observed.
After
After
After
Energy

Before
Chemical compounds are coloured because
they absorb visible light.
UV/visible
In general, even organicSpectroscopy
compounds that are
colourless will absorb UV light.

Absorption of visible light

Where has the energy that

was within the photons gone to ?
In UV/visible spectroscopy the energy of the absorbed photon is used to
drive the molecule into an excited electronic state.

In the excitation the energy of the whole molecule increases.

After

n
Before it o
rp ∆ E
Energy

s o
Ab
This overall change is typically due to promotion of a single
electron from a lower to higher energy orbital. The energy of the
transition depends on the gap between the two orbitals.
In organic compounds which have only single bonds between
the atoms the excitation energy is very high- lies in deep UV.

This excitation gives a dramatic decrease in bond order due to

excitation from

a bonding to an anti-bonding orbital

With increasing conjugation, the decreasing
energy gap is reflected by absorption at longer
wavelengths.
 The structures of many coloured compounds
show they are very extensively conjugated.

beta-Carotene

COOH O H
HOOC
H N O
N
trans-Crocetin
H2N N N
O O
Xanthopterin

OMe OMe
16,17-DimethoxyViolanthrone
Substituents added to the compound may alter the energy of
the orbitals which e- is excited from or to.

Auxochromes: substituents that alter the wavelength or

intensity of the absorption due to the chromophore
O NH2 O NH2

ORANGE PURPLE

O O OH

O NHCH3

BLUE

O NHCH3
Changes in chemical composition can give rise to
pronounced colour changes since this can
dramatically alter the energies of the orbitals involved
in the transitions e.g. pH indicators.

Phenolphthalein O
O
O
-2H
+

O O

OH
HO O-

colourless pink
Methyl orange
H3C
N red
H3C
N N
SO3-
H+

H3C
N H
H3C +
N N orange-yellow
SO3-
 The basic laws of photochemistry:

1. The Grotthuss-Draper law: Only those radiation which are absorbed by the
reacting system are effective in producing chemical change.

But the reverse of this law is not always true i.e. the system on absorbing light may
or may not result into a chemical reaction. In many cases, the absorbed light is
converted into the kinetic energy of the absorbing molecules and thereby only heat
effects are produced.

2. Laws of photochemical equivalence or Einstein law: Each light absorbing molecule

in a photochemical reaction absorbs only one quantum of light which causes the
activation.

∆ E = NAhν
It is conventionally known as one Einstein of energy.
 The Quantitative Picture
• Transmittance:
I0 I
T = I/I0 (power in) (power out)
• Absorbance:
A = -log10 T = log10 I0/I
B(path through sample)

• The Beer-Lambert Law:

A = ε bc
Where the absorbance A has no units, since A = log10 I0 /I
ε is the molar absorbtivity with units of L mol-1 cm-1
b is the path length of the sample in cm
c is the concentration of the compound in solution, expressed in mol L-1 (or M, molarity)
How Do UV spectrometers work?
Rotates, to
achieve scan
Two photomultiplier
inputs, differential
voltage drives amplifier.
Matched quartz cuvettes
Sample in solution at ca. 10-5 M.
System protects PM tube from
stray light
D2 lamp-UV
Tungsten lamp-Vis
Double Beam makes it a
difference technique
 Types of Electronic Transitions
Energy absorbed from UV radiation promotes an electron from highest occupied
molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO)

σ σ *
π π *

n π * n σ *
 σ - σ * Transitions (120-200
nm)
• An electron in a bonding σ orbital is
excited to the corresponding antibonding
orbital. The energy required is large. For
example, methane (which has only C-H
bonds, and can only undergo σ − σ *
transitions) shows an absorbance
maximum at 125 nm. Absorption maxima
due to σ − σ * transitions are not seen in
typical UV-Vis spectra (200 - 700 nm)
 n − σ * Transitions
• Saturated compounds containing atoms
with lone pairs (non-bonding electrons)
are capable of n − σ * transitions. These
transitions usually need less energy than
σ − σ * transitions. They can be initiated
by light whose wavelength is in the range
150 - 250 nm. The number of organic
functional groups with n − σ * peaks in the
UV region is small.
Methanol λ max = 183 nm ε = 500
Trimethylamine λ max = 227 nm ε = 900
 n − π * and π − π *

Transitions
• Most absorption spectroscopy of organic
compounds is based on transitions of n or
π electrons to the π * 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 π electrons.
 n − π * and π − π *

Transitions
(continue)
• Molar absorbtivities from n − π * transitions are
relatively low because the electrons in the n-
orbital are situated perpendicular to the plane of
the π bond and hence the probability of the
jump of an electron is very low and range from 10
to100 L mol-1 cm-1 .
π − π * transitions normally give molar
absorbtivities between 1000 and 10,000 L mol-1
cm-1 .
 The corresponding absorption band in UV-Visible spectrum may be
designated as follows:

• R band (group type, Radikalartig) originated from n - π * transition. єmax < 100
Example:
Acetone
λ max 279 nm, єmax <100

Hypsochromic shift (blue shift) with an increase in solvent polarity

• K band (conjugation band, Konjugierte) form π - π * transition. High єmax ( > 104)
Example:
Dienes, Acetophenone
B band (Benzene band, Benzenoid bands) from the π - π * transition of Benzene.
Broad band with fine structure between 230 – 270 nm. This band can be
used to identify aromatic compound.
• E band (Ethylenic bands) also from π - π * transition of ethylenic
band in benzene
E1 band and E2 bands of benzene occur near 180 and 200 nm
respectively and their molar absorptivity varies between 2000 and
14000.
 Important useful terms in UV-Vis spectroscopy
• The absorbing groups in a molecule are called chromophores

• A molecule containing a chromophore is called a chromogen

• An auxochrome does not itself absorb radiation, but can enhance the
absorption. If an auxochrome is attached to a C=C a bathochromic effect is
observed and if to a double bond where n electron are available (C=O) a
hypsochromic effect is observed.

• Bathochromic shift – red shift

• Hypsochromic shift – blue shift

• Hyperchromism – an increase in absorption

• Hypochromism – a decrease in absorption

• Isobestic Point: A point common to all curves produced in the spectra of a

compound. e.g. phenol-phenolate conversion as a function of pH can
demonstrate the presence of the two species in equm by the appearance of
an isobestic point in the spectrum.
 Solvent effect
• The solvent in which the absorbing species is dissolved
also has an effect on the spectrum of the species.
• Peaks resulting from n −π * transitions are shifted to
shorter wavelengths (blue shift) with increasing solvent
polarity. This arises from increased solvation of the lone
pair, which lowers the energy of the n orbital.
• The reverse (i.e. red shift) is seen for π − π *
transitions.
 Choice of solvent:
The solvent should be of high purity, generally referred as “spectrograde”

A good solvent should be transparent over the desired range of wavelength. Usually
solvents which do not contain conjugated system are most suitable for running the
Uv-visible spectrum.

The fine structure of an absorption band depend on the polarity of the solvent. A non-
polar solvent does not form hydrogen-bond with the solute and the spectrum of the
Solute clearly approximate to its spectrum in the gaseous state.
In a polar solvent hydrogen bonding forms a solute-solvent complex and the fine
structure may disappear.

A solvent should be chosen so that it does not react chemically with the sample.

N O O

CF3

Coumarin 153 (C-153)

Solvents for UV (showing high
energy cutoffs)

Water 210 THF 220

CH3C≡ N 210 CH2Cl2 235

C6H12 210 CHCl3 245

Ether 210 CCl4 265

EtOH 210 benzene 280
Hexane 210 Acetone 330
MeOH 215 Various buffers for
HPLC, check before
Dioxane 225
using.
 Woodward-Fieser rules for substituted dienes
(i) Parent acyclic diene 217 nm

(ii) Parent heteroannular diene 214 nm

(iii) Parent homoannular diene 253 nm

Increment for substitution
(iv) Alkyl group or ring residue (if the alkyl group is attached to +5 nm
two double bonds, count it twice)
(v) Exocyclic double bond (effect is two-fold if bond is exocyclic +5 nm
to two ring)
(vi) Double bond extending conjugation substituents on vinyl +30 nm
carbons (for each one)
(vii) Halogen (-Cl, -Br) + 5 nm

(viii) -OR, -O-(acyl) (-O-COR) + 6 nm, 0

nm
(ix) S-(alkyl) +30 nm
(x) -NRR- +60 nm
 The effect of steric hindrance to coplanarity

Woodward rules give reliable result only for those compounds in which there is no
strain around the chromophore.

Longer the conjugation, higher will be the absorption maximum and larger will be the
value of extinction coefficient. If in a structure the π electron system is prevented from
achieving coplanarity, there is a marked shift in the absorption maximum and ε max ..

This departure is due to steric crowding which distorts the geometry of the chromophore.
Hence the conjugation is reduced by reduction in the π orbital overlap.
 Study of charge transfer complex:

The formation of charge transfer complexes occurs between molecules which, when
mixed, allow transfer of electronic charge through space from an electron rich molecule
to an electron-deficient molecule.

The bond formation between the molecules occurs when filled π orbitals (or non-bonding
orbital) in the donor overlap with the depleted orbitals in the acceptor resulting in the
production of new MO.

The transition between these newly formed orbitals are responsible for the new
absorption band observed in the charge transfer complex.

The electron transfer from the donor to the acceptor is more complete in the excited
State than in the ground state and the wavelength of absorption is correlated with
electron affinity of the acceptor and the ionization potential of the donor.
The Benesi-Hildebrand method is a mathematical approach used in the determination
of the equilibrium constant K and stoichiometry of nonbonding interactions. This method
has been typically used to study reaction equilibriums that form 1:1 guest-host
complexes, where the guest and host are reactants, as shown below.

To observe one-to-one binding between a single host (H) and guest (G) using UV/Vis
absorbance, the Benesi-Hildebrand method can be employed. The basis behind this
method is that the acquired absorbance should be a mixture of the host, guest, and
the host-guest complex.

With the assumption that the initial concentration of the guest (G0) is much larger
than the initial concentration of the host (H0), then the absorbance from H0 should
be negligible.
(G0>>H0)
The absorbance can be collected before and following the formation of the HG complex.
This change in absorbance (ΔA) is what is experimentally acquired, with A0 being the
initial absorbance before the interaction of HG and A being the absorbance taken at
any point of the reaction.

Using the Beer-Lambert law, the equation can be rewritten with the absorption
coefficients and concentrations of each component

Due to the previous assumption that [G]0 >> [H]0, one can expect that [G] = [G]0.
Δε represents the change in value between εHG and εG.

A binding isotherm can be described as "the theoretical change in the concentration

of one component as a function of the concentration of another component at constant
temperature." This can be described by the following equation:
By substituting the binding isotherm equation into the previous equation, the
equilibrium constant Ka can now be correlated to the change in absorbance due
to the formation of the HG complex.

Further modifications results in an equation where a double reciprocal plot can be

made with 1/ΔA as a function of 1/[G]0. Δε can be derived from the intercept while Ka
can be calculated from the slope.
Limitation:
Conjugation, Color, and the
Chemistry of Vision
Colored organic compounds have extended
conjugated systems
• “UV” absorptions extend into the visible
region
∀ β -Carotene has λ max = 455 nm
– When white light strikes β -carotene wavelengths in
the blue region are absorbed while the yellow-orange
colors are transmitted to our eyes
Conjugation, Color, and the
Chemistry of Vision
β -carotene is converted in the human body to
11-cis-retinal, an essential molecule for vision
 Conjugation, Color, and the Chemistry of Vision

In the rod cells of the eye 11-cis-retinal is converted into

rhodopsin, a light-sensitive substance
When light strikes rhodopsin, trans-rhodopsin
(metarhodopsin II) is produced, which is accompanied
by a change in geometry
The change in geometry causes a nerve impulse to be
sent through the optic nerve to the brain, where vision
is perceived
Metarhodopsin II is then recycled back into rhodopsin
Conjugation, Color, and the
Chemistry of Vision

The cis-trans change in bond geometry accompanying vision

 Solvent effect (cont)
• The reverse (i.e. red shift) is seen for π − π * transitions.
This is caused by attractive polarisation forces between
the solvent and the absorber, which lower the energy
levels of both the excited and unexcited states. This effect
is greater for the excited state, and so the energy
difference between the excited and unexcited states is
slightly reduced - resulting in a small red shift.
• This effect also influences n −π * transitions but is
overshadowed by the blue shift resulting from solvation of
lone pairs.