Instrumentation:

Principles, working
and application of IR
Spectroscopy
• SMIC604
• Unit 3.1.i
• Snehal Dabhade
• Reference: Bioinstrumentation By VeenaKumari
 Background
• Energy of Molecules= Electronic Energy + Vibration Energy + Rotational Energy
• absorption of infrared light by a molecule results in transition to higher levels of
vibration
• the bonds between atoms can be considered as flexible springs, illustrating the
constant vibrational motion within a molecule.
• Bond vibrations can thus be either stretching or bending (deformation) actions.
• Theory predicts that a molecule with n atoms will have a total of 3n-6 fundamental
vibrations (3n-5, if the molecule is linear): 2n-5 bending, and n-1 stretching modes
 IR region: 0.8 µm ( 800
nm) to 1000 µm ( 1 mm)

Near IR: 0.8-2 µm Middle IR: 2-15 µm Far IR: 15-1000 µm

Most of the analytical applications are confined to middle IR region because the absorption of organic
Molecules are high in this region.
• When the energy in the form of infrared ration is applied then it causes vibration between the
atoms of the molecules and when Applied IR frequency = Natural frequency of vibration, the
absorption of IR radiation takes place and peak is observed.
• Different functional groups absorbs characteristic frequencies of IR radiation and hence gives
characteristic peak.
• Therefore, the IR spectrum of a chemical substance is a finger print of a molecule for its
identification.
 Principle
• The technique is based upon the simple fact that a chemical substance shows marked selective
absorption in the infrared region.
• After absorption of IR radiations, the molecules of a chemical substance vibrate at many rates of
vibration, giving rise to close-packed absorption bands, called an IR absorption spectrum, which
may extend over a wide wavelength range.
• Various bands will be present in IR spectrum that will correspond to the characteristic functional
groups and bonds present in a chemical substance.
• Thus, an IR spectrum of a substance is a fingerprint for its identification.
 SINGLE-BEAM INFRARED
•
SPECTROPHOTOMETER
In the single-beam system, the radiation is emitted by the source through the sample and then
through a fixed prism and a rotating Littrow mirror. Both prism and Littrow mirror select the
desired wavelength and then allow it to pass on to the detector. The detector measures the
intensity of radiation after it passes through the sample. Knowing the original intensity of radiation,
one can measure how much radiation has been absorbed. By measuring the degree of absorption
of wavelengths, the spectrum of the sample can be obtained.

Nichrome
alloy coil as
a source
Disadvantages The various disadvantages of a single-beam spectrophotometer are as follows:
1. This type of instrument has a basic disadvantage in that the intensity of the emission of the
radiation source varies from point to point in IR absorption spectrum; therefore, the resulting
spectrum is considerably deformed. The necessary correction by the continuous variation of slit
is cumbersome.
2. When the sample is analyzed in solution, the bands of solvent appear in the spectrum. In this
case, the spectrum of the sample is obtained by subtracting the spectrum of the from the
resultant spectrum, the former must be recorded under identical conditions (thickness of layer,
etc.) In order to overcome the above-mentioned difficulties, a double-beam spectrophotometer
is used.

3. Veerakumari, L. Bioinstrumentation (p. 333). MJP Publishers. Kindle Edition.

DOUBLE-BEAM INFRARED
SPECTROPOTOMETER
• Radiation source The source of radiation in a typical infrared spectrometer is a small ceramic rod,
heated electrically in the range 1100–1800ºC and is made of either silicon carbide (Glowbar) or
Nernst filament (a high-resistance, brittle element composed of mixture of a sintered oxides of
zirconium, thorium and cerium held together by a binding material).
• The radiation is divided into two beams, one of which passes through the sample while the other
functions as a reference beam. The reference and the sample beams are then passed alternately
into a monochromator at very short intervals by means of a rotating mirror.
• Absorption cells and sample preparation The cells generally employed are made up of rock salt or
potassium bromide (glass and quartz cells are unsuitable as these materials themselves absorb IR
radiation). A compound (0.55 mg) may be examined in solution in a suitable minimum absorbing
solvent (CCl4, CHCl3, etc.) as a liquid film. In the case of solids which are not soluble in these
solvents the compound can be examined as a mull in nujol or as a pellet obtained by pressing the
sample in a hydraulic press.

Nujol is a brand of mineral oil that plays a significant role in infrared (IR)
spectroscopy, particularly when analyzing solid samples. It is primarily used to
create a mull, which is a thick suspension of the sample in Nujol. This process
allows for the effective collection of IR spectra from solids, which can be
challenging due to their physical properties.
• Monochromator The pulse beam enters the monochromator through an entrance slit and is
dispersed by a grating or by a Littrow mount prism. In the monochromator, the emergent beams
are sorted out into individual wavelengths by means of a sodium chloride prism, which is
transparent to infrared radiation throughout the range 4000–650 cm–1. Lithium fluoride has more
favourable dispersion properties than sodium chloride at high wave numbers but is not transparent
below 1000 cm–1. In order to extend the operating range below 650 cm–1 prisms made up of
potassium bromide (transparent up to 400 cm–1) and caesium iodide (transparent up to 200 cm–1)
are also used. In high resolution rating instruments, a filter is used to reject radiation of unwanted
orders.
• Detector, Amplifier and Recorder The pulsating single beam now emerging through the slit is a
narrow band consisting of only a very few frequencies. After dispersion, the beams are focused
alternately at each particular wavelength throughout the spectral range, by means of a mirror
system on to the detector, usually a sensitive fast thermocouple. The signals from this are amplified
electronically. The spectrum, which is really a measure of the difference in intensities of the
reference and sample beams throughout the wavelength range, is recorded on a special graph
paper mounted on a rotating drum.

• Veerakumari, L. Bioinstrumentation (p. 335). MJP Publishers. Kindle Edition.

 • Detectors are of the heat recognition type.
• The Golay cells contains gas or liquid whose expansion is registered when energy is absorbed.
• Thermal detectors such as thermocouples can also be employed.

A Golay cell consists of a gas-filled

enclosure, typically filled with xenon gas,
which is sealed within a metal cylinder.
The interior of the cylinder contains an
infrared absorbing material that absorbs
incoming IR radiation. When this radiation
is absorbed, it heats the gas inside the
enclosure, causing it to expand. This
expansion leads to an increase in pressure,
which subsequently deforms a flexible
diaphragm or membrane located at one
end of the cylinder.
The deformation of the diaphragm results
in changes in light reflection off its surface,
which is detected by a photodiode. The
motion of the diaphragm produces a
change in signal on the photodiode that
correlates with the intensity of the
 when two dissimilar metals
are joined together and
subjected to a temperature
difference, an electromotive
force (EMF) is generated. This
EMF can be measured and is
proportional to the
temperature difference
between the junctions of the
two metals.
 Instrumentation:
Principles, working
and application of
AAS &AES (Flame
photometry)
SMIC604
Unit 3.1.ii
 ATOMIC SPECTROSCOPY
• The science of atomic spectroscopy has yielded three techniques for analytical use namely atomic
emission, atomic absorption, and atomic fluorescence.
• In atomic spectroscopy atoms have to be volatilized either in a flame or electrothermally in an
oven. In this state, the elements will readily emit or absorb monochromatic radiation at the
appropriate wavelength.
• Usually nebulizers (atomizers) will be used to spray the standard or the test solution into the flame
through which light is passed.
• Alternatively the light beam is passed in an oven through a cavity containing the vaporized material.
• The wavelengths emitted from excited atoms may be identified using a spectroscope, spectrograph,
or a direct reading spectrophotometer that use the human eye, a photographic plate or a
photoelectric cell, respectively as detectors.
• Molecules give rise to band spectra, but atoms yield clearly defined line spectra.
• In atomic emission spectroscopy (AES), these lines can be observed as light of a particular
wavelength (colour). Conversely, black lines can be observed against a bright background in atomic
absorption spectroscopy (AAS). T
• In a spectrum of an element, the absorption or
emission wavelengths are associated with
transitions that require a minimum of energy
change.
• In order for energy changes to be minimal,
transitions tend to occur between orbitals close
together in energy terms.
• For example, excitation of a sodium atom and its
subsequent relaxation gives rise to emission of
orange light (‘D-line’) due to the transition of an
electron from the 3s to the 3p orbital and return.
• Electron transitions in an atom are limited by the
availability of empty orbitals.
• Filling orbitals with electrons is subject to two
major rules: • one orbital can be occupied with a
maximum of two electrons; and the spins of
electrons in one orbital need to be paired in an
antiparallel fashion (Pauli principle).
• Together, these limitations mean that emission
and absorption lines are characteristic for an
individual element.
 spin quantum number is a
fundamental property that describes the
intrinsic angular momentum of
particles, electrons

• The Pauli exclusion principle is a

fundamental concept in quantum
mechanics that describes the behavior of
fermions, which are particles with half-
integer spins. Formulated by Austrian
physicist Wolfgang Pauli in 1925, this
principle states that no two identical
fermions can occupy the same quantum
state simultaneously within a quantum
system. This principle is crucial for
understanding the structure of atoms and
the behavior of matter at a microscopic
level.
 ATOMIC EMISSION SPECTROSCOPY
• In emission spectroscopy, a sample is excited by absorbing thermal or electric energy and the radiation
emitted by the excited sample is studied for both qualitative and quantitative analysis.

• Most of the spectroscopic techniques are related to molecules but emission spectroscopy is related to atoms.
Therefore, this technique is used as a method of elemental metal analysis.

• With it, all metallic elements can be identified and quantitatively determined in very low concentrations, as
can the metalloids such as arsenic, silicon and selenium.

• Solids, liquids or gases can be analyzed quite easily by this method. But most of the samples are solids or
evaporated solutions.

• Liquid samples are occasionally analyzed whereas gaseous samples are rarely analyzed. If proper precautions
are taken, the method can be used for quantitative analysis of about seventy elements at concentration level
as low as 1 ppm.
 Principle of AES
• If an atom is in the ground state, its electrons are present in the lowest
permitted energy levels.
• When electric or thermal methods excite the atom, its electrons move
from the inner orbital to the outer orbital.
• The excited electrons rapidly emit a photon of energy and occupy the
orbital with the lowest energy or ground state.
• The emitted radiation from the excited atoms in the form of discrete
spectral lines forms the basis of emission spectroscopy.
 Instrumentation of AES
• In emission spectroscopy, the sample is excited by thermal or electric methods and thus becomes the
source of radiation. Then, the radiation is passed through a monochromator to select the desired
wavelength and, finally, passed on to the detector, which measures the radiation for qualitative
analysis. The various individual components of the equipment are described below:
• Excitation sources The excitation sources in emission spectroscopy are the flame, direct or indirect
current arc and alternating current spark. Any excitation source used for emission spectroscopy must
accomplish the following process
1. The sample must be vaporized.
2. It must be dissociated into atoms.
3. The electrons in the atoms must be excited to higher energy levels above the ground state.
4. It should be capable of exciting atoms of all the elements of interest.
5. It should provide sufficient line intensity so as to detect these lines within detection limit.
6. It should provide reproducible excitation conditions from sample to sample.

• Veerakumari, L. Bioinstrumentation (p. 355). MJP Publishers. Kindle Edition.

Fuel Oxidant Temperature deg.cel.
Natural gas Air 1700
Natural gas oxygen 2700
Acetylene Air 220
Acetylene Oxygen 3200
Acetylene Nitrous Oxide 3400
Hydrogen Oxygen 2800

Table 11.4 Temperatures of commonly used fuels and oxidants

in flames
• Nebulizer The nebulizer passes a stream of air over a
capillary tube whose other end dips into the solution under
test. In indirect injection systems, large droplets will not
remain in the hottest part of the flame for long time, and
hence are allowed to settle in a cloud chamber. Combustion
of air and natural gas gives a temperature of 1500ºC which is
adequate for sodium determination. Calcium is better
analyzed at 2000–2500ºC, magnesium and iron require
2500ºC obtained from air/acetylene gas mixture.
• Monochromators In emission spectroscopy both prism and grating monochromators are used. The
function of the monochromator is to separate the various lines of a sample’s emission spectrum.
• Detectors Two types of detectors are widely used—photomultipliers and photographic plates or
films. For qualitative analytical studies, photographic plates or films are used. Photomultipliers are
used for quantitative analysis.
• Emission spectra may be examined by means of an optical arrangement that will identify
component frequencies and their intensities. This optical arrangement is known by several names.
For example, it is termed as a spectroscope if it involves a visual device; it is termed as a
spectrograph if it records many wavelengths simultaneously on a photographic plate or film; it is
termed as a spectrometer if it scans a spectrum. Except spectroscope, both spectrograph and
spectrometer determine intensities.
 ATOMIC ABSORPTION
SPECTROSCOPY
• Since its introduction by Alan Walsh in the middle of the 1950s atomic absorption spectroscopy has
been improved to become the most powerful technique for the determination of trace metals in
liquids. This method provides the total metal content of a sample and is almost independent of the
molecular form of the metal in the liquid.
 Principle AAS
• The absorption of energy by ground state atoms in the gaseous state forms the basis of atomic absorption
spectroscopy.
• When a solution containing metallic species is introduced into a flame, the vapour of the metallic species will
be obtained.
• Some of the metal atoms may be raised to an energy level sufficiently high to emit the characteristic radiation
of the metal—a phenomenon that is utilized in the familiar technique of emission flame photometry.
• But a large percentage of the metal atoms will remain in the non-emitting ground state. These ground-state
atoms of particular elements are receptive of light radiation of their own specific resonance wavelength.
• Thus, when a light of this wavelength is allowed to pass through a flame having atoms of the metallic species,
part of that light will be absorbed and the absorption will be proportional to the density of the atoms in the
flame.
• Thus, in atomic absorption spectroscopy, the amount of light absorbed can be determined. Once this value of
absorption is known, the concentration of metallic element can be determined because the absorption is
proportional to the density of the atoms in the flame.

.
 Instrumentation- AAS
• The principle of the instrumentation is similar to other spectroscopic absorption methods.
• Light of a certain wavelength (produced by a special kind of lamp), which is able to emit the
spectral lines corresponding to the energy required for an electronic transition from the ground
state to an exciting state, is allowed to pass through the flame.
• Meanwhile, the sample solution is aspirated into the flame. Before it enters the flame, the solution
gets dispersed into a mist of very small droplets, which evaporate in the flame to give first the dry
salt, and then the vapour of the salt.
• At least a part of this vapour will be dissociated into atoms of the element to be measured.
• Thus, the flame possesses free unexcited atoms which are capable of absorbing radiation from an
external source, when the radiation corresponds exactly to the energy required for a transition of
an element from the ground electronic state to an upper excited level.
• Then the unabsorbed radiation from the flame is allowed to pass through a monochromator, which
isolates the exciting spectral lines of light source. From the monochromator, the unabsorbed
radiation is sent to the photo detector, which is then recorded, the output of which is amplified
and measured on a recorder. Absorption is measured by the difference in the transmitted signal in
the presence and absence of the test element.
• Radiation source The radiation source for absorption spectrophotometer should emit stable,
intense radiation of the element to be determined, usually a resonance line of the element.
• A hollow cathode lamp is used as the radiation source.
• The spectral lines produced by the hollow cathode lamp are so narrow that they are completely
absorbed by the atoms.
• Each hollow cathode lamp emits the spectrum of that metal which is used in the cathode. For
example, copper cathode emits the copper spectrum, zinc cathode emits the zinc spectrum and so
on.
• In atomic absorption spectrophotometer, gaseous discharge lamps are also used. These are called
as arc lamps. Gaseous discharge lamps contain an inert gas at low pressure and a metal or metal
salt. These lamps are useful for the alkali metals, zinc, cadmium and mercury.
• Structure and Functioning of HCLs
An HCL consists of a glass tube containing a
cathode made from the element of interest,
an anode, and an inert filler gas, typically a
noble gas such as argon or neon. When a
high voltage is applied across the electrodes,
the buffer gas ionizes, creating a plasma.
This plasma accelerates ions towards the
cathode, where they collide with its surface
and sputter off atoms. These sputtered
atoms are then excited by collisions within
the plasma and subsequently emit photons
as they return to lower energy states.
• The emitted light contains spectral lines
characteristic of the element used for the
cathode. This property allows for selective
detection of specific elements when light
from the HCL passes through a sample
solution in an AAS setup.
• Chopper:A rotating wheel is interposed between the hollow cathode lamp and the flame. This
rotating wheel is known as chopper and is interposed to break the steady light from the lamp into
an intermittent or pulsating light. This gives a pulsating current in the photocell. There is also
steady current caused by light, which is emitted by a flame. But only the pulsating (or alternating)
current is amplified and recorded and, thus, the absorption of light will be measured without
interference from the light emitted by the flame itself.

• A pulsating current is a direct current (DC) that has regular variations in

magnitude.
• Production of the atomic vapour Before the liquid sample enters the burner, it is first converted
into small droplets. This method of formation of small droplets from the liquid sample is called
nebulization.
• A common method of nebulization is by the use of gas moving at high velocity, called pneumatic
nebulization.
• The Beckman total consumption burner is commonly used in atomic absorption measurements.
The fuel and oxidizing gases are passed through separate passages to meet at the opening of the
base of the flame.
• The flame breaks up the liquid sample into droplets which are then evaporated or burnt, leaving
the residue which is reduced to atom.
• Total consumption burners do use oxygen, with hydrogen or acetylene, and give very hot flames.
However, total consumption burner is noisy and hard to use. The efficiency of this burner is not
good. Hence a pre-mixed burner is used. A typical

• Veerakumari, L. Bioinstrumentation (pp. 360-361). MJP Publishers. Kindle Edition.

• In the pre-mixed burner, a mixture of the sample (liquid) and pre-mixed gases (C2H2+O2) is
allowed to enter the base, M and then to the region A. From the region A, the unburnt
hydrocarbon gaseous mixture and liquid droplets enter the region B. In the region B, the liquid is
evaporated leaving a residue. This residue is burnt in the regions C and D to produce atoms.
• Monochromators The function of a monochromator is to select a given absorption line from spectral
lines emitted from the hollow cathode. Prism or gratings are most commonly used as
monochromators to select a particular wavelength of light. When the cathode in the hollow cathode
lamp is made up of transition metals, the emission spectrum from the hollow cathode is so
complicated that high dispersion is essential. For such cases, large dispersion and high resolving
monochromators are advantageous for resolving spectra.
• Detectors For atomic absorption spectroscopy, the photomultiplier tube is most suitable. In the
photomultiplier tube, there is an evacuated envelope, which contains a photocathode, a series of
electrodes called dynodes and an anode. The photocathode is fixed to the terminal of the power
supply. As soon as a photon strikes the photocathode, an electron is dislodged and the photon is
accelerated to dynode 1, resulting in the liberation of two or more electrons. Thus, the current
multiplied at each dynode and the resultant electron current is received by the anode to produce
much larger photoelectric current, which goes to the external amplifier and read-out system.
• Amplifier The electric current from the photomultiplier detector is fed to the amplifier, which
amplifies the electric current many times.
• Read-out device In most of the atomic absorption measurements, chart recorders are used as read-
out devices. In some atomic absorption measurements, digital read-out devices are also used.

• .