Infrared Spctroscopy

Infrared radiation describes electromagnetic radiation at frequencies and

energies lower than those associated with visible light. (0.8-25mm)

The IR region of the electromagnetic spectrum is subdivided into three parts, namely, the
near-, mid-, and far-infrared, given as wavenumbers (i.e., wavelength/cm, units cm-1)

Far infrared (400-10 cm-1) lies adjacent to the microwave region of the electromagnetic
spectrum, is lowe energy and can be used in rotational spectroscope

Mid-infrared (4000-400 cm-1) is used to study fundamental molecular vibrations

Near-infrared (14000-4000 cm-1) is of high energy and can be used to generate overtones or
harmonic vibrations
IR spectroscopy depends on compounds absorbing energy – the major principles are
essentially the same as those of UV/Visible spectroscopy
An important fundamental difference lies in the fact that absorbed energy causes
vibrational excitation of bonds in a compound as opposed to electronic transitions
resulting from absorbed uv/viible light.

IR spectroscopy relies on the specific frequencies at which chemical bonds vibrate or

rotate corresponding to the energy levels applied to a compound.

Bonds can be excited by IR radiation to cause bond ‘ stretching’ or bond ‘bending’ e.

g., bands arise due to C=O stretching, C-H stretching etc.
Illustration of vibrational modes in IR spectroscopy. (Adapted from Silverstein
et al. Spectrometric Identification of Organic Compounds, 7th edn, 2005.)
Stretching or bending of bonds can be further classified into various
vibrational modes.

In the case of stretching, the modes can be either ‘symmetrical’ or ‘asymmetrical’

Bending modes: scissoring, rocking, twisting and wagging

Moderately sized compounds contain groups such as –CH2 or –NH2, which will
possess these different vibrational modes

Even simple molecules have many bonds, each of which can stretch and bend
independently, resulting in complex spectra
For IR, asymmetry gives much stronger vibrations and symmetric vibrations give rise to
only weak signals

Also for molecules to absorb IR light there must be a change in dipole moment
 Bonds are considered as springs – the behaviour of these molecular springs
approximately follows Hooke’s law of elasticity

Hooke’s law relates the strain on a body (spring) to the force (load or mass)
causing the ‘strain’.

In essence, molecular bonds follow this linear relationship where the extension of the
bond (or spring) is directly proportional to the force applied to the bond.
The following equation correlates vibrational frequency with bond strength and
atomic mass

Where n is vibrational frequency (Hz); c is the speed of light (3x 108m/s); k is the
force constant of the bond ( bond strength, units Nm-1), m is the reduced mass

Reduced mass relates to the relationship of the masses (atoms) at either end
of the molecular spring, termed m1 and m2, where reduced mass is defined as
(m1xm2/(m1 + m2)
e. g., in a C-H bond, the masses are C=m1 and H=m2
Considering the previous equation and remembering that energy also relates to
frequency (E= hn), a number of important points arise:
 Energy is directly proportional to bond strength

 Bond stretching requires more energy than bond bending, and as such bond
stretching absorbs shorter wavelength, higher frequency radiation than bond
bending
 When exciting vibration in bonds, single bonds require (and absorb) less
energy than double bonds, which require (and absorb) less energy than triple
bonds

 Energy is inversely proportional to reduced mass, and thus, the smaller the
reduced mass of a bond, the greater the energy (and frequency) required for
vibration. For example, C-H has a smaller reduced mass than C-C and therefore
stretching in induced at a higher frequency
  As the mass of the atom bonded to carbon increases (i.e., C-H to C-C), the
reduced mass increases, less energy is absorbed and the wavenumber
decreases

The more the energy absorbed by the bond, the higher the frequency and faster
the vibration

IR spectroscopy data (%T vs. wavenumber) gives important information on the

presence and characteristics of bonds in a given biomolecule as a result of
absorbance
The basic setup of an IR spectrometer is essentially similar to that of a
UV/visible spectrophotometer

 The IR source is an electric heated filament, which emits a continuous range

of frequencies (thus wavelengths and wavenumbers) in the IR region of the
electromagnetic spectrum

 The filament is commonly a Nernst filament(comprising Zr, Th and Ce oxides)

or Globar (silicon carbide)

 The emitted beam is passed through the sample in a cell (container) of

defined path length (10-2 cm for solutions) and IR is absorbed by the sample

 Similar to UV/visible spectroscopy, IR spectroscopy measures the intensity

reading of a reference (I0) and a sample (I) to generate %T.
 The sample cell is constructed from optically clear sodium chloride (not
plastic/glass or quartz) or KBr.

 The IR beam passes through the sample cell and emerges passing through a
series of slits and mirrors (to enhance resolution) and ultimately forms a
collimated beam that is passed through a monochromator that disperses the
beam into different wavelengths (light separation) and the detector which is a
thermocouple, convert radiant energy (heat) into an electrical current
The primary measures in IR spectroscopy are %T and wavenumber

Sample preparation is an important aspect of IR spectroscopy

There are a range of approaches depending on whether the sample to be analysed is solid,
liquid or gas

 A solid sample is typically prepared by crushing the sample with a mulling/dispersing

agent such as Nujol(mixture of hydrocarbons) and applying a thin layer of the mull to the
sample plate/cell

 An alternative method is to grind solid sample with KBr powder (purified salt) which s
then crushed in a mechanical die press to generate a transluscent KBr disk (the sample
plate/cell)
 A gas sample is introduced into a cylindrical sample cell (of inactive IR material, e.g.,
KBr or NaCl) that has a regulated inlet or outlet port to control gas flow into the cell
during the experiment.

 A liquid sample is introduced between the two plates (of inactive IR material e.g., KBr)
comprising the sample cell, and usually the liquid sample is prepared using an
anhydrous solvent such as chloroform as water can destroy the sample plate.

The chosen method of sample preparation can affect the spectra (position of peak
absorptions) and caution is required in data interpretation

In a typical IR spectrometer, the sample and reference are run in parallel, being
present in the instrument at the same time.

The spectrum (%T) is recorded over the range 4000-400 cm-1 generating dips as
opposed to absorbance peaks of UV/visible spectra
Interpretation of IR spectra

(1)The intensity of the peaks must be of sufficient magnitude and peaks should be
clearly visible and resolved

(2)Other factors include (i) purity and concentration of sample being analysed

(3)Solvent used for preparation of sample and reference

The IR spectra generated can be divided into four principal regions relating to molecular
vibrations, distinguished on the basis of wavenumbers (cm-1)

These all lie in the IR region of the electromagnetic spectrum and are 4000-2500 cm-1,
2500-1900 cm-1 and 1900- 1500 cm1 and 1500 cm-1 and below (fingerprint region)

4000-2500 region: area where bonds to hydrogen usually absorb – that is low reduced
mass of X-H, where X may be C, N, O, S

2500-1900 region: where triple bonds usually absorb e.g., C=X, where X may be C or N

1900-1500 region: C double bonds e.g., C=X, where X may be C, N or O. plus N=O

Fingerprint region : many bonds absorb, particularly single bonds e.g., C-O, C-N, C-C
Due to complexity in interpretation of data in the fingerprint region, it is preferentially
used for direct comparison with pure known compounds