Infrared Spectroscopy

• Atoms in a molecule do not maintain fixed

positions - they vibrate back and forth.
• The bond length is an average value.
• A diatomic molecule, e.g. H-Cl can only
undergo a STRETCHING vibration.
• More complex molecules exhibit both
STRETCHING and BENDING vibrations.
• Vibrational motion is excited within a single
electronic state by infrared light.

Vibrations of bonds involving HYDROGEN are very

significant as atoms of low mass move a lot more in
comparison to atoms of higher mass.
Simple Harmonic Oscillator
Treat a diatomic molecule as two masses joined by a spring
Hooke’s Law
re
The force to compress or extend
a spring is:

F(r) = – k (r - re)
m1
m2
Force Constant Equilibrium length
Actual length

Units of force constant are: N m-1

Strong spring - large force constant

Weak spring - small force constant

Diatomic molecule force constants

Strong bonds have large force constants.

Molecule k/N m-1 Eb/kJ mol-1 Eb = bond dissociation

H2 509 436 energy.
HCl 478 431 Single bonds
Cl2 320 242
N2 2241 945 triple bond

Potential energy in the spring, V(r)

V(r) = 1 k (r – re)2
2
Vibrational frequency, n
The vibrational frequency is given by:

m m
n = 1 k
 Hz  = m + m2
1
kg
2 1 2

µ is the reduced mass

SI Units
• Hz (or s-1) for n
• N m-1 for k
• kg (per molecule) for µ

We frequently use masses in g mol-1 (e.g. H=1, C=12, O=16 …)

 = mm 1+mm2 u kg
Where u is the unified mass
1 2 constant u = 1.66  10-27 kg
 Vibrational frequency - wavenumbers, cm-1
n = 1/l and c = lnso n = n / c The SI unit of wavenumber is m-1.
But cm-1 is more common.
n/m n/cm 100
The vibrational frequency n = 1 k
in cm-1 is given by: 200 c  cm-1

The frequency is:

1. directly proportional to the force constant
2. inversely proportional to the reduced mass
Molecule k/N m-1 µ/kg n/cm-1
H2 509 8.3110-28 4159
HCl 478 1.6210-27 2890
Cl2 320 2.9110-26 557
N2 2241 1.1610-26 2331
Example

Suppose that the C=O group in a peptide bond can be regarded as

isolated from the rest of the molecule. Given that the force constant
of the bond in a carbonyl group is 908 N m-1, calculate
the vibrational frequency of (a) 12C=16O and (b) 13C=16O.
Energy levels
Classically the harmonic oscillator
can have any energy.
Quantum mechanically it only has
discrete equally spaced energy
levels given by:
Ev = (v + 12 ) hn v = 0, 1, 2, 3…

Selection rule
Absorption of light only occurs for ∆v = ± 1
This means that each transition has the same energy change.
Interaction with light
1. Light has an oscillating electric field.
2. The molecule must also possess an oscillating electric field (at
the same frequency).
3. Photon energy must equal the difference between energy levels.
Oscillating dipole
1. Heteronuclear diatomics have a dipole due to the uneven
electron distribution around each atom.
2. The dipole is the product of the charge  distance apart.
3. Bond vibrations change the distance and hence the dipole
oscillates. This creates an oscillating electric field.
d+ d+ d+ d+
d+
d- d- d- d-
d-
max min
 Infrared inactive
1. Homonuclear diatomics (H2, N2, Cl2 etc.) do not have a dipole
and cannot absorb light in the IR to excite bond vibrations. (The
vibrational motion of homonuclear diatomics can be probed
using RAMAN spectroscopy).

2. In many molecules, some vibrations are IR inactive, some are

IR active. The requirement is a net oscillating dipole, e.g. CO2
d- d+ d- d- d+ d-

Dipoles cancel Dipoles cancel

d- d+ d- d- d+ d-

Dipoles cancel Dipoles DO NOT cancel

CO2 symmetric stretch CO2 asymmetric stretch

inactive active
Characteristic Vibrations of H2O - all IR active

symmetric stretch bend asymmetric stretch

n1 = 3657 cm-1 n2 = 1595 cm-1 n3 = 3756 cm-1
Number of vibrational modes

Linear molecule with n atoms - number modes = 3n–5

e.g. CO2 n = 3 so number vib modes = 33–5 = 4

Non-linear molecule with n atoms - number modes = 3n–6

e.g. CH4 n = 5 so number vib modes = 35–6 = 9

IR spectra are complex

The number of vibrational modes increases
very rapidly with n, PLUS many of these
vibrations may occur at the same frequency
and so not all of the possible bands are seen
as independent absorptions. Fullerene C60
174 vib modes
Number of vibrational modes

Linear molecule with n atoms - number modes = 3n–5

e.g. CO2 n = 3 so number vib modes = 33–5 = 4

Non-linear molecule with n atoms - number modes = 3n–6

e.g. CH4 n = 5 so number vib modes = 35–6 = 9

IR spectra are complex

The number of vibrational modes increases
very rapidly with n, PLUS many of these
vibrations may occur at the same frequency
and so not all of the possible bands are seen
as independent absorptions. Fullerene C60
174 vib modes
Stretching and bending vibrations in organic molecules

Bending vibrations can be:

Stretching vibrations can be: (i) scissoring (in-plane)
(i) symmetric (ii) rocking (in-plane)
(ii) asymetric (iii) wagging (out-of-plane)
(iv) twisting out of plane

Infrared INACTIVE vibrations

• Not all vibrations result in absorption bands.
• For a vibration to cause IR absorption the dipole moment of
the molecule must change when the vibration occurs.
IR spectra of Organic/Biological molecules

• Each stretching and bending vibration of a bond is associated

with radiation whose frequency exactly matches the frequency
of the vibration of the bond.
• Different functional groups, e.g. C–C, C=C, C=O, N–H …
will have different characteristic frequencies
• Therefore, by determining the frequencies of light (IR) that are
absorbed by a particular compound we can determine what
kind of bonds it has.

This is not as easy as it sounds

 IR spectra of Organic molecules
Gas Phase Infrared Spectrum of Formaldehyde, H2C=O

3n–6 = 34–6 = 6
6 vibrational modes

The spectra of simple

molecules can often
be fully assigned
Wavenumber / cm-1

IR spectra are often shown as % Transmittance vs Wavenumber.

So, ZERO absorption is 100% transmittance.
An absorption band appears as a trough in the spectrum. i.e. the
spectrum looks ‘upside down’.
 IR spectra of Organic molecules
% Transmittance

IR spectrum of 1-propanol

CH2
bending

C-C stretch
OH stretch Backbone
CH3 stretch stretches
312–6 = 30
Wavenumber 30 vibrational modes

1. The liquid/solid state usually produces broader lines.

2. The spectra of larger molecules are very complicated and
it is often not possible to assign all the peaks involved.
Lactic Acid

Functional group Region Fingerprint Region

Backbone
CH3 stretches stretches

OH stretches carboxyl
stretches

The fingerprint region is an area of complex overlaps where it is

impossible to assign all (any?) of the bands. It can act as a unique
‘fingerprint’ for a given molecule.
Spectral regions in the infrared

Middle IR spectrum can also be roughly divided into:

1. FUNCTIONAL GROUP REGION,  = 4000 - 1200 cm-1

2. FINGERPRINT REGION,  = 1200 - 400 cm-1

• It takes more energy to stretch a bond than bend it

• Therefore stretching bands are found in the
4000-1200 cm-1 (functional group region)
• Bends are typically observed in the fingerprint region
Functional group frequencies

There are extensive tables and charts available to aid in identifying

IR spectral features. It’s as much an art as a science!
Functional group frequencies
 Applications of IR to Protein structure
O
Amide bond of peptides has many vibrational Amide I
C
modes (at least 10 IR bands) these bands are N
designated amide I, amide II, etc.
Amide II
H
The frequency of Amide I depends upon the environment of
the bond and as such can be used to learn about the protein structure.

Amide I Amide II
Protein Amide I: 1690-1600
Protein Amide II: 1575-1480
 Amide Band Assignments

Amide A is 95% due to the the N-H stretching vibration. It is very sensitive to the
strength of the hydrogen bond.
Amide I is primarily governed by the stretching vibrations of the C=O (70-85%) and
C-N groups (10-20%).
Amide II derives mainly from in-plane N-H bending (40-60%). The rest arises from
the C-N (18-40%) and the C-C (about 10%) stretching vibrations.
Amide III, V are very complex bands dependent nature of side chains and hydrogen
bonding - only of limited use for the extraction of structural information.
Amide I band

Its frequency depends upon whether the peptide bond is in the

-helix, -sheet, or random configuration
Wavenumber /cm-1

-helix 1650

-sheet 1632, 1685

random coil 1658

Each band is well resolved so if protein contains all three

configurations 4 bands will be seen.