INFRARED
SPECTROSCOPY
INTRODUCTION
The infrared portion of the electromagnetic spectrum is divided into
three regions; the near-, mid- and far- infrared, named for their relation to
the visible spectrum. The far-infrared, approximately 400-10 cm-1
(1000–30 μm), lying adjacent to the microwave
region, has low energy and may be used for rotational spectroscopy. The mid-infrared,
approximately 4000-400 cm-1 (30–1.4 μm) may be used to
study the fundamental vibrations and associated rotational-vibrational structure. The higher
energy near-IR, approximately 14000-4000 cm-1 (1.4–0.8 μm)
can excite overtone
or harmonic vibrations. Photon energies associated with this part of the infrared (from 1 to 15 Kcal/mole)
are not large enough to excite electrons, but may induce vibrational excitation
of covalently bonded atoms and groups. 
Figure 1: Electromagnetic
spectrum showing the infrared regions
PRINCIPLE
The absorption of
electromagnetic radiations in the infrared regions results in vibrational and
rotational transitions of the bonding atoms in the molecule leading to
asymmetrical charge distribution. Non linear molecules may undergo vibrational
motions like stretching and deforming. The energy requirement of stretching
vibrations (either symmetrical or asymmetrical) are higher and occur at higher
frequencies than deforming or bending (either in plane; scissoring or out of
plane; rocking i.e. wagging and twisting) vibrations.
The absorption of infrared
radiations doesn't cause excitation of electrons but induces vibrations of
bonding atoms. The vibrations which bring about alteration in dipole moment or
displacement of charge can be detected. The other types of vibrations are
studied by Raman spectroscopy.
Figure 2: Mode of
vibrations of molecules in infrared lights
Figure 3: Electromagnetic spectra of IR-Spectroscopy
Figure 3: Electromagnetic spectra of IR-Spectroscopy
The fundamental vibrations
are those which correspond to V0 – V1 transitions. The
intensity of a particular absorption depends on the dipole moment of the
molecule in the ground state and vibrational excited state. Higher is the
difference in dipole moments, greater will be the intensity of absorption. The
IR spectra are observed only in heteronuclear molecules since homonuclear
molecules have no dipole moment. For obtaining an infrared spectrum,
electromagnetic radiation of increasing wavelength is passed through the sample
and percent transmittance or absorbance is measured. The energy of vibrations
is quantized. The IR spectrum is functional groups in molecules are highly specific
and this characteristic spectrum facilitates identification of compounds
according to frequency units rather than wavelength.
INSTRUMENTATION
A
beam of infrared light is produced and split into two separate beams. One is
passed through the sample, the other passed through a reference which is often
the substance the sample is dissolved in. The beams are both reflected back
towards a detector, however first they pass through a splitter which quickly
alternates which of the two beams enters the detector. The two signals are then
compared and a printout is obtained. The IR instruments have following parts;
1.
IR source: The source of light is generally an
incandescent lamp having output maxima at around 2 cm. However their energy
shows a sharp decline (15% of maximum at 15 cm). Neonest-Glober and nichrome
alloy wire lamps have been used for this purpose.
Figure 4: Diagrammatic
representation of IR-spectroscopy
2. Sample containers: The
sample containers used in spectrophotometer such as Glass, quartz, silica
cuvettes are impermeable to infrared lights. Non-covalent materials are
transparent to infrared light. Thus sodium chloride
or common salt plates are used commonly as sample containment. A number of
other salts such as potassium bromide
or calcium fluoride
are also used. The plates are transparent to the infrared light and will not
introduce any lines onto the spectra. Some salt plates are highly soluble in
water, so the sample and washing reagents must be anhydrous
(without water). The window materials for IR-spectroscopy; NaCl
6000 – 600 cm–1
KBr
6000 – 450 cm–1
CsI
6000 – 200 cm–1
Polyethylene
< 600 cm–1
3. Sample Preparation: Gaseous
samples require little preparation beyond purification, but a sample cell with
a long pathlength
(typically 5-10 cm) is normally needed, as gases show relatively weak absorbance.
Liquid
samples can be sandwiched between two plates of a high purity salts and measure
the absorbance.
Solid
samples can be prepared in two major ways. The first is to crush the sample
with a mulling
agent (usually Nujol; which is hydrocarbon oil, with strong absorption around 2900 and
1400 cm–1) in a marble
or agate mortar, with a pestle.
A thin film of the mull is applied onto salt plates and measured. The second
method is to grind a quantity of the sample with a specially purified salt
(usually potassium bromide)
finely (to remove scattering effects from large crystals). This powder mixture
is then crushed in a mechanical die press
to form a translucent pellet through which the beam of the spectrometer can
pass.
It is important to note that spectra obtained from different sample
preparation methods will look slightly different from each other due to
differences in the samples' physical states.
The last technique is the Cast Film technique. This technique is
used mainly for polymeric compound. Sample is first dissolved in suitable, non
hygroscopic solvent. A drop of this solution is deposited on surface of KBr or
NaCl cell. The solution is then evaporated to dryness and the film formed on
the cell is analyzed directly. Care is important to ensure that the film is not
too thick otherwise light cannot pass through. This technique is suitable for
qualitative analysis.
4. Reference: A reference is usually a
pure form of the solvent the sample is in. It is used for two reasons: i. This prevents fluctuations in the output
of the source affecting the data. ii. This
allows the effects of the solvent to be cancelled out.
5. Detector: The detector is a thermopile which comprises of
stack of thermocouple junction in an evacuated can having an infrared window.
USES AND APPLICATIONS
1.
Infrared spectroscopy is widely
used in both research and industry as a simple and reliable technique for
measurement, quality control and dynamic measurement. The instruments are now
small, and can be transported, even for use in field trials. With increasing
technology in computer filtering and manipulation of the results, samples in
solution can now be measured accurately (water produces a broad absorbance
across the range of interest, and thus renders the spectra unreadable without
this computer treatment.
2.
The main application of
infrared spectroscopy in biochemical investigations is in elucidation of
structure of purified biological molecules of intermediates size such as small
peptides, metabolic intermediates, drugs etc.
3.
It is employed for examining
the secondary structure of proteins. Polypeptides give intensity bands at 3290,
1650 and 1535 cm–1
providing clues about the secondary structure. E. g. In alpha helix, amide I
bands splits into two absorbance maxima of 1650 cm–1(strong one) and
1652 cm–1(medium one) intensity. While amide II give weak intensities
at 1516 and 1546 cm–1 only.
4.
By measuring at a specific
frequency over time, changes in the character or quantity of a particular bond
can be measured. This is especially useful in measuring the degree of
polymerization in polymer
manufacture. Modern research machines can take infrared measurements across the
whole range of interest as frequently as 32 times a second. This can be done
whilst simultaneous measurements are made using other techniques. This makes
the observations of chemical reactions and processes quicker and more accurate.
5.
Infrared spectroscopy has been
highly successful for applications in both organic and inorganic chemistry.
Infrared spectroscopy has also been successfully utilized in the field of
semiconductor microelectronics: for example, infrared spectroscopy can be
applied to semiconductors like silicon, gallium
arsenide, gallium nitride, zinc
selenide, amorphous silicon, silicon
nitride, etc.
6. Isotope
effects: The
different isotopes in a particular species may give fine detail in infrared
spectroscopy. For example, the O-O stretching frequency of oxyhemocyanin is experimentally determined to be 832
and 788 cm-1 for ν (16O-16O) and ν (18O-18O)
respectively.
LIMITATIONS OF
IR-SPECTROSCOPY
- It will measure only sample molecules with a dipole moment.
- It is very difficult to reproduce exact results in IR-spectroscopy because of changes in pathlength & changes in concentration.
- If absorption is not in the 4000-400 cm-1 range it will not show on spectrum graph in IR-spectroscopy.
- Absorption is too weak to be observed.
- Absorptions can be too close to each other.
- Additional weak bands of vibrations are observed.
