BP701T. INSTRUMENTAL METHODS OF ANALYSIS UNIT-II

IR SPECTROSCOPY
UNIT-II
Prepared And Presented By
Mr. Sandip R. Bhoi
Assistant Professor
KVPS’s Institute of Pharmaceutical Education. College, Boradi

INTRODUCTION
➤ Infrared or vibrational spectroscopy is the study of the absorption of Infrared
radiations resulting in vibrational transitions.
➤ Infrared spectroscopy is an important analytical technique for determining the
structure of both organic & inorganic compounds.
➤ Lies in the wavelength range of 0.8-1000 µm
➤ Between visible and microwave regions.
➤ Atoms in a molecule do not remain in fixed positions but vibrate about their mean
positions.
➤ On absorption of IR transition from ground vibrational level to excited vibrational
level give rise to closely packed absorption spectrum.

Mid-IR region-2.5-25 µm
 Vibrational rotational region.
 Based on absorption of specific wavelengths of mid infrared light by a molecule.
 Absorption, reflection & emission spectra are employed for both qualitative and
quantitative analysis.
Fingerprint region
 Falls on the right side of the spectrum.
 Complicated series of absorption bands due to interacting vibrating modes.
 Each different compound produces a different pattern of troughs and bands.
 Absorption pattern is unique for each molecular species.

Types of molecular vibrations
a. Stretching vibrations.
b. Bending vibrations
Stretching vibrations
 Here the distance between two atoms increases or decreases but the atoms remain in the same
bond axis.
 Symmetrical molecules like O=C=O are not IR active because no change in dipole moment is
observed upon stretching vibrations.
 Stretching vibrations are of two types:
 1. Symmetrical stretching: When two bonds increase or decrease in length.
 2. Asymmetrical stretching: When one bond length increases, the other decreases.

 Bending vibrations
 Involve movement of atoms that are attached to a common central atom, such that there is a
change in the bond axis & bond angle of each atom without change in their bond lengths.
 Bending vibrations require less energy & occur at longer wavelengths than stretching
vibrations.
 Also called deformation vibrations.
 Types of bending vibrations
1. In-plane vibrations
a) Scissoring b) Rocking
2. Out plane vibrations
a) Wagging b) Twisting

1. In-plane vibration
a) Rocking: in-plane bending of atoms occurs where they swing back & forth to the central atom.
b) Scissoring: 2 atoms connected to the central atom move towards and away from each other.
2. Out-plane vibrations
a) Wagging: two atoms oscillate up and below the plane to the central atom.
b) Twisting: one atom moved up the plane while the other down the plane concerning the central
atom.

SAMPLE HANDLING
➤Sample handling is considered as an important technique in the infrared spectroscopy.
➤The samples used in IR spectroscopy can be either in the solid, liquid, or gaseous state.
➤ Samples of different phases have to be handled differently.
➤ A common point is that the material containing the sample must be transparent to IR
radiations.
SAMPLING OF SOLIDS
Generally, 4 techniques are employed for preparing solid samples:
1. Solids run in solution.
2. Solid Films.
3. Mull technique.
4. Pressed pellet technique.

1. SOLIDS RUN IN SOLUTION
• Solids may be dissolved in a non-aqueous inert solvent and a drop of this solution is placed
on an alkali metal disc and the solvent is allowed to evaporate, leaving a thin film of solute (or
the entire solution is placed in a liquid sample cell) which is then mounted in a spectrometer.
• If the solution of solid can be prepared in a suitable solvent then the solution is run in
concentration of cells for liquids.
• Some solvents used are chloroform, carbon tetrachloride, acetone, Cyclohexane, etc.
Precautions:
• Solute chemical interaction with the solvent must be taken into consideration, especially for
compounds having the property of H-bonding.
• The solvent should not absorb in the studied range.

2. Solid Films.
get salt plate
from the
desiccator
Put about 50
mg of solid
into a beaker,
vial, or test
tube
add a couple of
drops of
solvent
(usually
acetone or
methylene
chloride) to
dissolve the
solid.
Place a drop of
this solution on
a salt plate.
Allow the
solvent to dry;
you should
observe a thin
solid film on
the plate.

Place the plate in the V-shaped sample holder
inside the instrument. Note that you use only
one salt plate in this procedure, not two plates
as when running a thin liquid film.

3. Mull technique.
Materials Required:
Gather the necessary
materials, including
the solid sample,
Nujol (mineral oil),
clean and polished IR-
transparent salt plates
(such as KBr), an
agate mortar and
pestle, a mounting
card, and a spatula.
Plate Preparation:
Ensure that the IR
plates are clean and
free from any residual
material. It is
advisable to rinse the
plates with hexanes to
remove any
contaminants before
use.
Sample and Oil Mixing:
Take a small portion of
the solid sample and add
approximately 10% of
the sample volume
worth of Nujol. Grind
the mixture in the agate
mortar and pestle until it
forms a homogeneous,
transparent mull with no
visible particles.

 Application of the Mull: Once the mull is prepared, add a drop of the mull to one IR plate.
Place the second plate on top of the drop and give it a quarter turn to evenly coat the surface
of the plates.
 Data Acquisition: Insert the sandwiched plates into the spectrometer to acquire the desired
IR spectrum. Always handle the plates with gloves and avoid exposure to water or solvents
during the process.
• This method is good for qualitative analysis but not for quantitative analysis.
4. Pressed pellet technique.
• In this technique a small amount of finely ground solid sample is intimately mixed with
about 100 times its weight of powdered Potassium bromide, in a vibrating ball mill.
• This finely ground mixture is then pressed under very high pressure (25000 p sig) in
evaluable die or Minipress to form a small pellet (about 1-2 mm thick and lcm in diameter).
• The resulting pellet is transparent to IR radiation and is run as such.

SAMPLING FOR LIQUIDS:
• Liquid samples taken.
• Put it into rectangular cells of KBr, NaCl, etc.
• IR spectra were obtained.
• Sample thickness such that transmittance lies between 15-20 % i.e. 0.015-0.05 mm in
thickness.
• For double beams, matched cells are generally employed.
• Protect from moisture.

SAMPLING OF GASES:
•The gas sample cell is similar to the cell for liquid samples and is made of KBr, NaCl & so
on.
• the cells are larger, usually they are about 10 cm long, but they may be up to 1m long.
• Multiple reflections can be used to make an effective path length as long as 40 cm so that
constituents can be determined.
•Gas must not react with the cell windows or the reflecting surfaces.
• Gas analyses are performed with IR but the method is not commonly used because of its lack
of sensitivity.
• Moisture can be avoided.
•It is strong absorption bands at 3701cm-1 and 1625cm-l may interfere with the analysis.

FACTORS AFFECTING VIBRATIONS
1. Coupled vibrations
2. Fermi resonance
3. Electronic effects
4. Hydrogen bonding
COUPLED VIBRATIONS
• An isolated C-H bond has only one stretching vibrational frequency whereas the methylene
group shows two stretching vibrations, symmetrical and asymmetrical.
• Because of mechanical coupling or interaction between C-H stretching vibrations in the
CH2 group.
• Asymmetric vibrations occur at higher frequencies or wave numbers than symmetric
stretching vibrations.
• These are known as coupled vibrations because these vibrations occur at different
frequencies than that required for an isolated C-H stretching.

FERMI RESONANCE
 The coupling of two fundamental vibration modes produces two new modes of vibration,
With frequencies higher and lower than observed in the absence of interaction. Interaction
can also occur between fundamental vibrations and overtones or combination tone
vibrations; such interactions are known as Fermi Resonance.
 If two different vibrational levels. belonging to the same species. have nearly the same
energy.
 A mutual disturbance of energy may occur.
 Shifting of one towards lower and the other towards higher frequency occurs.
 A substantial increase in the intensity of the respective bands occurs.

 Example with Carbon Dioxide (CO ):
₂
1.Symmetrical Stretching Vibration:
1. Frequency: CO has a symmetrical stretching vibration that shows a
₂
band at 1337 cm ¹
⁻ in the Raman spectrum.
2.Bending Vibrations:
1. Frequency: CO also has two equivalent bending vibrations that
₂
absorb at 667.3 cm ¹
⁻ .
3.First Overtone:
1. Calculation: The first overtone of the bending vibration is 2 x 667.3
cm ¹ = 1334.6 cm ¹
⁻ ⁻ .
4.Fermi Resonance Occurs:
1. Interaction: The symmetrical stretching vibration at 1337 cm ¹
⁻ and
the first overtone of the bending vibration at 1334.6 cm ¹
⁻ interact

ELECTRONIC EFFECT
 Changes in the absorption frequencies for a particular group take place when the substituents in
the neighborhood of that specific group are changed.
It includes:
1. Inductive effect 2.Mesomeric effect 3. Field effect
INDUCTIVE EFFECT
• The introduction of the alkyl group causes the +I effect which results in the lengthening or the
weakening of the bond
• Hence the force constant is lowered and the wave number of absorption decreases.
• Let us compare the wave numbers of v (C=O) absorptions for the following compounds:
1750 cm-1.
1745 cm-1.
1715 cm-1.

MESOMERIC EFFECT
This effect can either make a bond longer or weaker, which lowers the frequency of
absorption.
1.Methyl Benzoate vs. Benzamide:
Methyl benzoate has a
carbon-oxygen bond
that absorbs at a higher
frequency (1730
cm ¹).
⁻
Benzamide has a nitrogen atom that
is less electronegative than oxygen.
This means the electron pair on
nitrogen is more available for
bonding, which makes the bond
weaker and absorbs at a lower
frequency (1693 cm ¹).
⁻

FIELD EFFECT
 A lone pair of electrons present on the atoms influence
each other through space interactions
and changes the vibrational frequencies of both groups.
 This effect is called as Field effect.
HYDROGEN BONDING
 Strong hydrogen bonds = longer bonds, lower frequency, broader bands.
 Weaker hydrogen bonds in amines = smaller shifts.
 Dilution helps distinguish between inter and intramolecular hydrogen bonds.

 Sources of IR radiation used in IR spectrophotometer
 1.Incandescent lamp
 This lamp is particularly used in near IR instruments. However, it is least preferred over
other sources as it has a low spectral emission.
 2. Nernst Glower
 It contains a hollow rod of rare earth oxides such as zirconia, yttria, and thoria.
 It is non-conducting at room temperature and requires heating by external means to bring it
to a conducting state.
 The glower is heated to a temperature within the 1000-1800° C range.
 It gives a maximum radiation of 7100 cm-1

 3.Globar source
 It is a rod prepared from centered Silicon carbide
 it is heated up to a temperature between 1300-1700°C
 it emits maximum radiation at 5200cm-1
 It has a disadvantage in that its radiation is less intense than Nernst glower
 4. Mercury arc lamp
 It is a device made up of a quartz-jacketed tube containing mercury vapor inside it at a
pressure greater than 1 atmosphere.
 It is highly effective in the far-IR region where the other sources of radiation fail to provide
continuous radiation.

Wavelength selectors
 They help in selecting continuous IR radiation in the desired wavelength region.
 They generally contain a chopper and a complex system of monochromators.
These monochromators are of two types:
1. Prismatic monochromator: They are made up of glass/quartz and coated with alkyl
halides.
A] Mono Pass (Radiation passes only once through the prism)
B] Double Pass (Radiation passes twice through the prism)
2. Grating monochromator: They are grooves made up of aluminium and provide better
dispersion of radiation than prisms.
A] Reflection gratings
B] Transmittance grating

Infrared spectrophotometry uses two types of detectors:
1.Thermal Detectors: These detectors measure the heating effect caused by infrared
radiation, which generates a potential difference proportional to the radiation amount.
Common thermal detectors include thermocouples, bolometers, thermistors, and Golay
cells.
2.Photo-Detectors:
 I. Thermocouples
 are the most commonly used detectors in infrared spectrophotometry.
 They consist of two metal strips joined at one end. These strips are made of different
metals and are welded with blackened gold foil.
 The welded joint (hot junction) is exposed to radiation, causing a temperature rise, while
the other joint (cold junction) is kept at a constant temperature.
 The temperature difference between the junctions generates a potential difference, which is
proportional to the amount of radiation falling on the hot junction.

 II. Bolometers
 detect infrared radiation by measuring changes in
electrical resistance due to temperature changes.
 They are part of a Wheatstone bridge circuit, with one
arm exposed to radiation and the other kept constant.
 When radiation hits the bolometer, the bridge becomes
unbalanced, causing an electrical current to flow through a galvanometer.
 The current’s magnitude indicates the radiation intensity.
 Bolometers have a response time of 4 milliseconds.

 III. Thermistors
 are similar to bolometers but are made from fused metallic oxides.
 They have a property where their electrical resistance decreases as the temperature
increases (negative thermal coefficient).
 This change in resistance helps measure the amount of infrared radiation
IV. Golay Cell (Golay Detector):
• Consists of a small metal cylinder.
• One end is closed by a blackened metal plate, the other by a metalized diaphragm.
• Cylinder is filled with non-absorbing gas like xenon.
• Infrared radiation heats the blackened metal plate.
• The heated plate causes gas expansion, moving the diaphragm.
• Diaphragm motion changes the output received by a phototube.

V. Pyroelectric Detectors in IR Spectroscopy:
• Function: Detect weak infrared radiation by generating electric outputs from changes in
thermal energy.
• Materials: Made from polar dielectrics that exhibit the pyroelectric effect when
temperature changes.
• Operation: No need for cooling or electrical biasing; works at room temperature.
• Sensitivity: High sensitivity to infrared radiation, making them suitable for various
applications.

 Photon Detectors:
 Widely used in the near-infrared region.
 Made of semiconductors like lead sulfide, lead telluride, or germanium.
 Non-conducting at lower energy states.
 Radiation raises them to a higher energy level, making them conductive.
 Conductivity produces a signal proportional to the radiation amount.
 Electrical resistance drops when exposed to radiation.
 Small voltage application results in a large current increase.
 Current can be amplified and displayed on a meter or recorder.

 Applications
 IR spectroscopy is applied for qualitative as well as quantitative analysis of drugs in the
Pharmaceutical industry.
 It is used for identification of drug substances
 It identifies the impurities present in a drug sample
 It helps in the study of hydrogen bonding both intermolecular and intramolecular type
 It is widely used in the study of polymers
 It helps determine the issue of Cis- Trans isomers present in a mixture of compounds
 It elucidates reaction mechanisms
 It is a great tool investigation of rotational isomerism
 It identifies functional groups present in any sample
 It estimates the relative stability of confirmation
 It distinguishes between Sis and trance
 It can predict the keto-enol tautomerism

 Introduction:
 During the 1980s Bowling Barnes, David Richardson, John Berry, and Robert Hood
developed an instrument to measure the low concentrations of sodium and potassium in a
solution.
 They named this instrument a Flame photometer.
 The principle of the flame photometer is based on the measurement of the emitted light
intensity when a metal is introduced into the flame.
 The wavelength of the color gives information about the element and the color of the flame
gives information about the amount of the element present in the sample.
 Flame photometry is one of the branches of atomic absorption spectroscopy.
 It is also known as flame emission spectroscopy.
 Currently, it has become a necessary tool in the field of analytical chemistry
 Flame photometers can be used to determine the concentration of certain metal ions like
sodium, potassium, lithium, calcium cesium, etc.
 In flame photometer spectra the metal ions are used in the form of atoms.

 Principle of Flame Photometer
 The alkali and alkaline earth metals (Group II) compounds dissociate into atoms when
introduced into the flame.
 Some of these atoms further get excited to even higher levels. However, these atoms are not
stable at higher levels.
 Hence, these atoms emit radiations when returning to the ground state. These radiations
generally lie in the visible region of the spectrum. Each of the alkali and alkaline earth
metals has a specific wavelength.

 Interferences
 The interferences can be of three types. They are:
1] Spectral interference: When two elements present similar spectra which are overlapping
each other and both emit radiation at the same particular wavelength it is known as spectral
interference / cation-cation interference / molecular spectral interference.
Example:
 Na and K mixtures interfere with each other.
 Al interferes with emission lines of Ca and Mg
2] Vaporization interference: This type of interference is caused by to presence of acids
which affects the dissociation with other metals. Also, high viscosity presents interferences
in the vaporization process affecting the overall atomization process.
3] Ionisation interference: High-temperature flames cause ionization of some of the metal
atoms present in both ground and excited states decreasing overall method sensitivity.

 1. Sample delivery system:
 The sample delivery system contains a sample holder and a nebulizer.
 A nebulizer is part of a sample delivery system in which the liquid droplets of
comparatively larger size are broken or converted into smaller sizes
 The process of conversion of a sample into a mist of very fine droplets through jets of
compressed gas is called nebulization.
 Hence, this part of the sample delivery system is called a nebulizer.
 2. Burner and flame
 Different types of burners are used to convert the fine droplets of sample solution into
neutral atoms which further due to the high heat or temperature of the flame are excited
finally these excited atoms emit radiation of characteristic wavelength and color.

 3. Filters and Monochromators:
 In flame photometry the wavelength and intensity of the radiation emitted by the element
are monitored. Hence a filter or monochromator is to be used in the instrument. A simple
flame The photometer contains a filter wheel containing several filters for elements like
Calcium, Lithium, Sodium, or Potassium and when a particular element has to be analyzed
a specific filter is Chosen.
 Similarly, the monochromators convert polychromatic light into monochromatic. Two types
of monochromators are generally used for this purpose.
 A] Prism: It is made up of quartz material and it is transparent over the entire region of
 measurement
 B] Grating: It employs grating which is a series of parallel straight lines cut into a plane
surface.

 4. Detectors
 Photomultiplier tube (PMT) detector is the most sensitive of all the detectors available
and expensive. The principle employed in this detector depends on the multiplication of
photoelectrons by the secondary emission of electrons. This multiplication is achieved by
using a photocathode and a series of anodes called is dynodes. The PMT can use up to 10
dynodes. These dynodes are maintained at 75-100V higher than the preceding dynode.
After each stage, the electron emission is multiplied by a factor of 4-5. The PMT can detect
very weak signals.
 The photovoltaic cell has a thin metallic layer coated with silver or gold which the app
says an electrode also has a metal base plate that acts as another electrode. The two layers
are separated by a semiconductor layer of Selenium. When light radiation falls on the
selenium layer it creates a potential difference between the two electrodes and causes flow
of current. The flow of current causes deflection of the galvanometer needle which depends
on the wavelength and intensity of radiation.

 5] Read out device The signal from the detector is shown as a response in the digital reader
device the readings are displayed as an arbitrary scale i.e. % flame intensity.

 The oxidants in flame photometers are mainly air, oxygen, or nitrous oxide. The
temperature of the flame depends on the ratio of fuel and oxidant.
 The processes occurring during flame photometer analysis are summarized below:
 Desolvation: Desolvation involves drying a sample in a solution. The metal particles in the
solvent are dehydrated by the flame and thus solvent is evaporated.
 Vaporization: The metal particles in the sample are also dehydrated. This also led to the
evaporation of the solvent.
 Atomization: Atomization is the separation of all atoms in a chemical substance. The metal
ions in the sample are reduced to metal atoms by the flame.
 Excitation: The electrostatic force of attraction between the electrons and nucleus of the
atom helps them to absorb a particular amount of energy. The atoms then jump to the higher
energy state when excited.
 Emission: Since the higher energy state is unstable the atoms jump back to the ground state
or low energy state to gain stability. This jumping of atoms emits radiation with a
characteristic wavelength. The radiation is measured by the photodetector.

 Applications
 Alkali and alkaline earth metals can be estimated by flame photometry.
 Many alkali and alkaline metals amount can be detected by flame photometry by using the
method of internal standard, method of standard addition, direct comparison method, and
calibration curve method. Examples of such quantifications include
(a)Determination of concentration of calcium in serum
(b)Determination of concentration of calcium, sodium, and potassium in urine
(c)Determination of the amount of sodium potassium, calcium, and magnesium in
intravenous fluid and oral rehydration salts.
(d) Assay of potassium chloride in syrup
(e)Determination of concentration of Lithium in serum for therapeutic drug monitoring.

ATOMIC ABSORPTION SPECTROSCOPY

 PRINCIPLE:
 The technique uses the principle that free atoms (gas) generated in an atomizer can absorb
radiation at a specific frequency.
 Atomic-absorption spectroscopy quantifies the absorption of ground-state atoms in the
gaseous state.
 The atoms absorb ultraviolet or visible light and make transitions to higher electronic
energy levels. The analyte concentration is determined by the amount of absorption.
 AAS is the most powerful technique for the analysis of trace elements.
 Approximately 70 elements can be analyzed by AAS.
 Determination of a single element in the presence of other elements is possible ( no need
for their separation)

 Interference :
 Any process that causes an error in determination is called interference.
 Interferent is the substance present in the sample, blank or standard solution which affects
the signal of the analyte.
 1) Chemical Interference:
 Because of the formation of stable compounds which cannot undergo decomposition at
flame temperature.
 Example Aluminum and magnesium form a thermally stable mixed oxide, thus low results
are obtained for magnesium in the presence of aluminum.
 Chemical interference affects the number of free atoms reaching the optical path to be
absorbed. Precipitation, viscosity, surface tension, and pH are some factors that cause
chemical interference

 2) Spectral Interference:
 When spectral lines overlap with each other then this kind of interference is observed.
 Absorption or emission of an interfering species either overlaps or lies so close to the
analyte band that resolution by the monochromator becomes difficult. In AAS spectral
interference is rare.

 3) Ionization Interference:
• High flame temperatures can cause atoms to ionize.
• Atoms with low ionization potential ionize, reducing the number of ground-state and
excited-state atoms.
• Adding easily ionizable elements like potassium (K) and sodium (Na) to the sample (1000
µg/L) helps counteract this effect.
 4) Background Absorption:
• Background absorption occurs when other species absorb light at the resonance
wavelength.
• This happens due to light scattering from small, un-volatilized particles in the flame.
• It’s a wavelength-dependent phenomenon that can cause positive errors in analysis.

1. Radiation source
Hallow cathode lamp:-
 Hallow cathode lamp consists of two electrodes i.e. anode and cathode.
 The anode is made up of tungsten, nickel, or zinc metal, while the cathode is
A hollow cylinder made up of metal to be analyzed.
 Both electrodes are enclosed in glass cylinders containing inert gas-like
Argon, Neon, etc.
 At the end of the hollow cathode lamp, there is a small window made up of
Quartz, Silica, or glass for transfer of radiation.
 Life depends on the current used during operation,
The average lifetime of HCL is two years.

 2. Chopper:
 A rotating wheel placed in between Hallow cathode lamp and the flame is known as
chopper. Function of chopper is to break the steady light from HCL in to intermittent light.
This gives alternating current in photocell.
 3. Automizer:
 Automizer convert sample solution in to atomic vapour.
 Important functions of flame
1) To evaporate solvent
2) To dissociate sample in to molecule
3) To provide ground state atoms for analysis
 Temperature of flame is kept low, because at high temperature ionization of atoms take
place which do not show absorption
 Types of Burners
 1) Total consumption burner 2) Laminar flow /Premix burner

4. Nebulizer:
 In nebulization sample is converted into a fine mist or droplets using a jet of compressed
gas.
 The flow carries the sample into the atomization region.
 Pneumatic Nebulizers :
 The liquid sample to be analyzed is sucked by a capillary with
high pressure by a gas moving at high velocity.
 This process is also called aspiration.
 Because of high-velocity sample breaks into fine
droplets (mist) and carries it to the atomization region

 5. Monochromator:
 Commonly used monochromators in AAS are prisms and gratings. Monochromator select
monochromatic light from polychromatic light emitted by a hollow cathode lamp.
 6. Detector:
 Photomultiplier tube Construction:
 It contains a photosensitive half-cylinder of metal which acts as a cathode.
 The inner surface of the cathode is coated with light-sensitive material.
 It consists of 9 dynodes which have a coating of cesium metal that emits several electrons.
 These electrons are collected by collecting electrodes (anode).
 This process is continued up to the 9th
dynasty. Emitted electrons are collected by the
collecting electrode and the current begin to flow. This current is amplified and measured
by a read-out device.
 Advantages: 1) It is very fast (response time is 10-9 seconds)
2) High sensitivity for U.V. and visible region.

 7. Amplifier: It amplifies the current from the photomultiplier tube.
 8. Read-out device: A chart recorder is the most common read-out device. Microammeter
is the other read-out device used in AAS.

 Applications of AAS:
 Qualitative Analysis:
 Different hollow cathode lamp is required for different elements. ( It can detect only
elements whose hallow cathode lamp is used) hence scope of AAS in qualitative analysis is
limited.
 Quantitative Analysis:
 A.A.S. is a powerful technique for the quantitative analysis of trace metals.
1) Concentration of unknown sample: Calibration
2) Simultaneous multicomponent analysis:
 By using a multicomponent Hallow cathode lamp, elements like Zn, Cd, Ni, Ca, Fe, Mn,
Cu, and Mg can detected in spectral regions 232 to 328 nm.

3) Analysis of biological material and food material:
 Trace metals from the biological system, Ni from vegetable oil, Cu from beer, and Na and
K from blood serum can be analyzed by AAS. It is also used to find impurities present in
food material.
 4) Determination of lead in petrol: Tetra ethyl lead ((C2H5 )4Pb)and tetramethyl lead
((CH3 )4Pb) are two anti-knocking agents used in petrol.

 Introduction:
 When an EMR or Light passes through a moderately stable suspension, a portion of incident
radiant energy is degenerated by Absorption, Refraction, and Reflection, and the remaining
portion of light gets transmitted.
 The Suspension particles have property scattering of light is termed the Tyndall effect
 Scattering of Light- changes the direction of light in multiple planes without changes in the
net radiating power of energy.

 Scattering of Light- changes the direction of light in multiple planes without changes in the
net radiating power of energy
 Scattering depends- a) No of Particles, b) the Dimension of particles, and c) the wavelength
of light
 Nephelometric and turbidometric methods depend on light scattering by particles suspended
in a liquid. The suspended particles have refractive index values different from the
suspending medium. The overall effect mimics the Tyndall effect.
 Nephelometry measures scattered light as a function of the concentration of suspended
particles where the concentration is less than 100 mg/liter.
 Turbidometry is the measurement of transmitted light as a function of the concentration of
suspended particles where the concentration is more than 100 mg/liter, which is a high
concentration of samples.

 Principle
 At lower concentrations of the suspension uniform scattering of particles is noticed. So the
intensity of scattered light is directly proportional to the concentration of solute. The
intensity of scattered light can be measured at 45°, 60°, 90° and 135°also.
 For higher-concentration suspensions scattering is nonuniform and light becomes scattered
in all possible directions. Hence, it is difficult to measure the intensity of scatter radiation at
specific angles. So, the intensity of transmitted light (that is unscattered) direction is
measured at 180°.
 Suspensions with lower concentration nephelometry and for higher concentration
turbidometry are utilized.

 3. Instrumentation
 There are separate instruments available as nephelometer and turbidimeter. Also, they are
combined to form a Nephloturbidometer. The following are the common instrumental
components:
1. Source of light
A tungsten lamp is used when a polychromatic light is necessary and a Mercury arc lamp is
used when a monochromatic light is necessary.
2. Filters and Monochromators: When polychromatic light is used filters and
monochromators are not required but when monochromatic light is necessary a filter or
monochromator is used. In turbidimeter blue filter of 530 nm is used and in a nephelometer
visible filter is used as a secondary filter.
3. Sample cells: Various shapes of sample cells are used in nepheloturbidimetry. They may be
cylindrical with a path length of 1 cm. Also, they may be rectangular cells but the cell walls
may be coated with black to avoid any reflection that may interact with detector response.
Cells can also be prepared as per need to measure the scattered light at different angles of 45°,
90°, 135° and 180°. These cells are made up of glass.

 Detectors: Photometric detectors like photovoltaic cells photomultiplier tubes are used in
such instruments. Photovoltaic cells or phototubes are used in turbidimeters. Whereas in a
nephelometer photomultiplier tubes are used as the scatter radiation is weak.

 Nepheloturbidimeter If is suspension is to be measured whose concentration is unknown
then at nepheloturbidimeter can be effectively used as it can detect both low-concentration as
well as high-concentration suspensions. It has two detectors one for measuring the scattered
light at 90° and the other one at 180° for transmitted light. The ratio of the response of two
detectors is displayed as nepheloturbidimetric units or NTUs which is proportional to the
ability of the suspension.

 Applications
 The nepheloturbidimeter can be effectively used for various purposes such as:
 Analysis of clarity of water
 Determination of carbon dioxide
 Determination of inorganic substances like phosphorus ammonia sulphate etc.
 For quantitative analysis of ions at ppm levels
 Analysis of petroleum products, sugar products, and clarity of citrus juices
 In turbidimetric titration

BP701T. INSTRUMENTAL METHODS OF ANALYSIS UNIT-II

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BP701T. INSTRUMENTAL METHODS OF ANALYSIS UNIT-II