Practical Analysis

FINAL YEAR B. PHARM.
SEM VII PHARMACEUTICAL ANALYSIS V
EXERCISE NO 1 Date:
Aim: To study principle, instrumentation and applications of

U. V. visible spectrophotometry.
References
1) Willard, H. H., Merritt Jr, L. L., Dean, J. A., & Settle Jr, F. A. (1988).
Instrumental methods of analysis,page no. 119, 148
2) Beckett A.H. and Stenlake, J.B. eds., 1988. Practical Pharmaceutical
Chemistry: Part II Fourth Edition (Vol. 2). A&C Black, page no. 275, 325
3) Skoog, D.A., Holler, F.J. and Crouch, S.R., 2017. Principles of instrumental
analysis. Cengage learning, page no 378, 411
PRINCIPLE
Spectroscopic analysis is a study of interaction of light or radiation with

matter. Spectroscopic methods refer to the measurement of the intensity of
radiation with a photometric transducer or other type of electric device. There
are two theories of nature of light which explain the different interactions of
radiations with matter.
Photon theory
According to photon theory, the light is made up of energy packets. These

energy packets are called photons. This theory helps us to understand the
process associated with the absorption & emission of radiation energy.
Electro-magnetic radiation theory
The properties of electromagnetic radiation are conveniently described by

means of a classical wave model, which embodies such characteristics as
wavelength, frequency, velocity & amplitude. Electromagnetic radiation is
conveniently represented as electric & magnetic field that undergo in phase
sinusoidal oscillation at right angle to each other & to the direction of
propagation.
SCES’S INDIRA COLLEGE OF PHARMACY, PUNE

FINAL YEAR B. PHARM. SEM VII PHARMACEUTICAL ANALYSIS V
DEFFINATIONS
Amplitude: It is the length of the electric vectors at a maximum in the wave.
Period: The time in seconds required for the passage of successive maxima or
minima through a fixed point in space is called period of radiation. (p)
Frequency: It is the no. of oscillations of the field that occurs per second & is
equal to1/p.
Wavelength: It is the linear distance between any 2 equivalent points on

successive waves.
Wave No.: It is the reciprocal of wavelength is cms unit
Constructive Interference: A maximum amplitude occurs when the two

waves are completely in phase; under these circumstances, the maximum
constructive interference is said to occur.
Destruction Interference: The resultant amplitude is smaller than the

amplitude of the component wave. This is called destruction interference.
λ max : The wavelength which is absorbed maximum by a molecule is called
as wavelength of maximum absorption. (λ max)
INTERACTION OF MOLECULE WITH RADIATION
The energy of the radiation in the visible range is generally: 36 to 72 kcal/mole

while that in the ultraviolet range goes as high as 143 kcal/mole. This energy
irradiated on the molecules can result in changes in the electronic nature of
the molecule i.e. changes between ground state and excited states of electrons
within the system. As a result, UV-visible spectroscopy is also known as
electronic spectroscopy.
Every time a molecule has a bond, the atoms in a bond have their atomic
orbitals merged to form molecular orbitals which can be occupied by electrons
of different energy levels. Ground state molecular orbitals can be excited to
anti-bonding molecular orbitals.

The electrons in a molecule can be of one of three types: namely σ (single

bond), π (multiple-bond), or non-bonding (n- caused by lone pairs). These
electrons when imparted with energy in the form of light radiation get excited
from the highest occupied molecular orbital (HOMO) to the lowest unoccupied
molecular orbital (LUMO) and the resulting species is known as the excited
state or anti-bonding state.
1. σ-bond electrons have the lowest energy level and are the most stable
electrons. These would require a lot of energy to be displaced to higher energy
levels. As a result these electrons generally absorb light in the lower
wavelengths of the ultraviolet light and these transitions are rare.
2. π-bond electrons have much higher energy levels for the ground state.
These electrons are therefore relatively unstable and can be excited more
easily and would require lesser energy for excitation. These electrons would
therefore absorb energy in the ultraviolet and visible light radiations.
3. n-electrons or non-bonding electrons are generally electrons belonging to

lone pairs of atoms. These are of higher energy levels than π-electrons and can
be excited by ultraviolet and visible light as well.
Most of the absorption in the ultraviolet-visible spectroscopy occurs due to π-

electron transitions or n-electron transitions. Each electronic state is well
defined for a particular system i.e. a double bond in 2-butene would have a
particular energy level for the π-electrons which when absorbs a specific (or
quantized) amount of energy would get excited to the π* energy level for the
electrons.

Selection rules for electronic transitions
π to π* transition is symmetry allowed - High intensity with ε more than 1000
n to π* transition is symmetry forbidden – low intensity with ε less than 100
Transition among electronic states of same spin multiplicity are allowed. If

there is change of spin multiplicity then it is forbidden. Therefore singlet to
singlet transition or triplet to triplet transition are allowed, but singlet to triplet
or triplet to singlet are forbidden.
BEER LAMBERT’S LAW
1) Beers law states that intensity of a beam of parallel monochromatic

radiation decrease exponentially with the no. of absorbing molecules ie
absorbance is proportional to concentration.
2) Lamberts law states that intensity of a beam of parallel monochromatic

radiation decreases exponentially as it passes through a medium of
homogenous thickness.
A combination of the two Law yields the Beer’s-Lamberts Law
Log I0
A= = abc
Log IT
Where;
I0 = Initial light intensity

IT = Transmitted light intensity

a=proportionality constant called absorptivity
b=thickness of solution/path length in cm.
c=concentration in gm/lit
Molar Absorptivity: Absorbance of light per unit path length (usually the
centimetre) and per unit of concentration in moles per litre is molar
absorptivity. It is a fundamental unit in spectrophotometry. Molar absorptivity
is a term used in chemistry to measure how a particular chemical absorbs light
at a particular wavelength. It is also known as molar extinction coefficient
denoted by 'ε'.
A = ε bC
Specific Absorbance: Another form of Beer-Lambert’s law proportionality
constant is the specific absorbance, which is the absorbance of a specific
concentration in a cell of a specific path length. The most common in
pharmaceutical analysis is the A1%
1Cm , which is absorbance of the solution of
1gm/100ml in a 1cm cell.

A = A1%
1Cm . b. C
Relationship between Molar absorptivity and Specific absorbance

ε = A1%
1Cm X molecular weight
Concept of Chromophore and Auxochrome in the UV spectroscopy

Chromophore is defined as any isolated covalently bonded group that shows a
characteristic absorption in the ultraviolet or visible region (200-800 nm).
Chromophores can be divided into two groups-
a) Chromophores which contain p electrons and which undergo pie to pie*
transitions. Ethylenes and acetylenes are the example of such chromophores.
b) Chromophores which contain both p and nonbonding electrons. They
undergo two types of transitions; pie to pie* and nonbonding to pie*. Carbonyl,
nitriles, azo compounds, nitro compounds etc. are the example of such
chromophores.

An auxochrome can be defined as any group which does not itself act as a
chromophore but whose presence brings about a shift of the absorption band
towards the longer wavelength of the spectrum. –OH,-OR,-NH2,-NHR, -SH etc.
are the examples of auxochromic groups.
UV CUT OFF WAVELENGTH OF SOLVENT
Every solvent has a UV-vis absorbance cutoff wavelength. The solvent cutoff is
the wavelength below which the solvent itself absorbs all of the light. So when
choosing a solvent be aware of its absorbance cutoff and where the compound
under investigation is thought to absorb. If they are close, chose a different
solvent. The following table provides an example of solvent cutoffs.
Solvent UV Cut off wavelength
Acetone 329 nm
Benzene 278 nm
Dimethylformamide
267 nm
(DMF)
Ethanol 205 nm
Toluene 285 nm
Water 180 nm
Hexane 211 nm
INSTRUMENTATION
Instruments for measuring the absorption of ultraviolet and visible
radiation are made up of one or more-
1 Sources
2 Wavelength selectors
3 Sample containers
4 Radiation transducers
5 Signal processors and Read-out devices.
1) Sources
Following are the requirements of the radiation sources

1. It must be stable
2. It must be of sufficient intensity for the transmitted energy to be
detected at the end of optical path
3. It must supply continuous radiation over the entire region
Radiation sources used are as follows:
Deuterium & hydrogen lamp: A continuous spectrum in ultraviolet
region is produced by electrical excitation of deuterium and or hydrogen
at low pressure. The mechanism by which a continuous spectrum is
produced involves initial formation of an excited molecular species
followed by dissociation of excited molecule to give two atomic species as
well as an ultraviolet photon.
Tungsten filament lamps: It is a common source of visible and near IR
radiation. The energy distribution of this source is temperature
dependent. It contains a small quantity of iodine within a quartz envelope
that houses the tungsten filament. Quartz allows the filament to be
operated at a temperature of about 3500˚K which leads to higher
intensities and extends the range of the lamp well into UV region.
Light emitting diodes: They are made up of gallium, aluminum,
arsenide, etc. They are used to shift the wavelength maximum anywhere
in the region of 375nm to 1000nm.
Xenon arc lamp: It produces intense radiation by passage of current
through an atmosphere of xenon. The spectrum is continuous over the
range between about 200-1000nm, with peak intensity occurring at
500nm.
2) Wavelength selectors
Most analysis require radiation that consists of a limited, narrow,
continuous group of wavelengths called a ‘band’. A narrow bandwidth
enhances the intensity of absorbance measurements. Ideally, the output
from a wavelength would be radiation of a single wavelength or frequency.
The effective bandwidth is the inverse measure of the quality of the
device, a narrower bandwidth representing a better performance. There
are 2 types of wavelength selectors.

A) Filters
B) Monochromators.
A) Filters:
 Interference filters: These rely on optical interference to provide
narrow bands of radiation.
 Absorption filters: These function by absorbing selected portions of
spectrum. They have effective bandwidth that range from 30 to 250nm.
Filters that absorb narrower bandwidths also absorb a significant
fraction of the desired radiation. For narrow bandwidths the fraction of
light transmitted by absorption filters is small.
B) Monochromators:
For many spectroscopic methods, it is necessary to be able to
continuously vary the wavelength of radiation over a broad range. This
process is called scanning of spectrum. Monochromators for U.V. Visible
and Infrared radiation are all similar in the mechanical construction in
the sense that they use slits, lenses, mirrors, windows and grating or
prisms.
Components
1. An entrance slit that provides a rectangular optical image.
2. A collimating mirror or lens that produces a parallel beam or
radiation.
3. A prism or a grating that disperses the radiation into its component
wavelengths.
4. A focusing element that reforms the image of the entrance slit and
focuses it on planar surface called focal plane.
5. Exit slit in the focal plane that isolates the desired spectral band.
Two types of dispersing elements are found in Monochromators:
 Prisms
 Reflection gratings.
Prism monochromator
Prisms have higher dispersion in the UV region. Prism monochromators
are favored in some instruments that are principally designed to work in

the far UV region. Most monochromators use gratings though. Some

monochromators have several gratings that can be selected for use in
different spectral regions.
Grating Monochromator
Grating monochromators are located within compartments of some UV
Visible instruments and are responsible for producing narrow bands of
radiation. There are five components found in most grating
monochromators: an entrance slit, a collimating lens or mirror, a
reflection grating, a focusing element, and an exit slit.
Monochromator slits
The slit of a monochromator play an important role in determining the

monochromators performance characteristics and quality. Slit jaws are
formed by carefully machining two pieces of metal to give sharp edges. Care is
taken to assure that the edges of the slit are exactly parallel to one another
and that they lie on the same plane.

The entrance slit of a monochromator serves as a radiation source, its image

is ultimately focused on the focal plane that contains the exit slit. If the
radiation source consist of a few discrete wavelengths, a series of rectangular
images appear on this surface as bright lines. A particular line can be brought
to focus on the exit slit by rotating the dispersing element.
If the entrance and exit slits are of the same width, in theory the image of the
entrance slit will just fill the exit slit opening when the setting of the
monochromator corresponds to the wavelength of the radiation.
Movement of monochromator setting in one direction or the other produces a

continuous decrease in emitting intensity, which reaches zero when the
entrance slit image has moved a distance equal to its full width.
Selection of slit width
The effective bandwidth of a monochromator depends on the dispersion of the

grating or prism as well as the width of the entrance slit and exit slit. Most
monochromators are equipped with variable slits so that effective bandwidth
can be changed. The use of minimal slit width is desirable when narrow
absorption or emission bands must be resolved. On the other hand, the
available radiant power decrease significantly when slits are narrowed and it
becomes more difficult to measure the power accurately. Thus wider slit
widths may be used for quantitative analysis.
Stray light: Stray Radiant Energy (SRE) or stray light is the measured
quantity of light that reaches the detector that is of a wavelength other than
that selected. Therefore, stray light causes the measured transmittance to be
erroneously high.
C) Sample containers
(1) The cells or cuvettes that hold the sample and solvent must be
constructed of a material that passes radiation in the spectral region of
interest.

(2) Quartz or fused silica can be used as material of construction of cuvette

as these materials are transparent in the UV-Visible region.
(3) Best cells minimize the reflection losses by having windows that are
perfectly normal to the direction of the beam.
(4) The most common path length for studies in UV and visible regions is
1cm.
(5) Special care must be taken to reproduce the position of cell with respect
to the beam, otherwise variations in path length and reflection losses at
the curved surfaces can cause significant errors.
(6) The quality of absorbance data depends critically on the way the cells
are used and maintained. Fingerprints, grease or other deposits on the
ways markedly alter the transmission characteristics of a cell. Thus, it
is essential to clean cells thoroughly before and after use.
D) Radiation Transducers
Transducers convert radiant energy into electrical energy. An ideal transducer

would have a high sensitivity, a high signal to noise ratio and a constant
response over a considerable range of wavelengths. In addition, it would
exhibit a fast response time and a zero output signal in the absence of
illumination. Finally the electrical signal (S) produced by ideal transducer
should be directly proportional to radiant power P.
S=KP
S = current /voltage output
K = calibration constant.
Many real transducers exhibit a small constant response known as ‘Dark

current’ in the absence of radiation. For these transducers, the response is
described
S=kP+Kd

K d = dark current which is usually constant over a short measurement

periods. Instrument with transducers that produce dark current are often
equipped with a compensating circuit that reduces K d to zero.
Types of radiation transducers
(a) Transducers which respond to heat

(b) Transducers which respond to photon/ radiation
All the photon transducers have an active surface that absorbs radiation. In
some types the absorbed energy cause emission of electrons and the
production of a photocurrent. In others, the radiation promotes electrons into
conduction, detection here is based on the resulting enhanced conductivity.
The thermal transducers respond to average power of the incident radiation.
Thermal transducers
The thermal transducers respond to average power of the incident radiation.

In thermal transducers, the radiation impinges on and is absorbed by a small
blackbody and a temperature rise is measured.
Photon transducers
Different photon transducers in use are as follows-
(1) Photovoltaic cell, in which radiant energy generates current at the

interface of semiconductor layer and a metal.
(2) Phototubes, in which radiation causes emission of electrons from a
photosensitive solid surface.
(3) Photomultiplier tubes, which contain a photo emissive surface as well
as several additional surfaces that emit a cascade of electrons when
struck by electrons from the photosensitive area.
(4) Photoconductivity transducers in which absorption or radiation by a
semiconductor produces electrons sand holes, thus leading to enhance
d conductivity.
(5) Silicon photodiodes, in which photons cause the formation of electron-
hole pairs and a current.

(6) Charge transfer transducers in which the charges developed in a silicon

crystal as a result of absorption of photons are collected and measured.
Barrier layer photovoltaic cell
It consist of a flat copper or iron electrode on which it deposited a layer of

semiconducting material such as selenium. The outer surface of
semiconductor is coated with a thin transparent metallic film of gold or silver,
which serves as a second electrode.
When radiation of sufficient energy reaches the semiconductor, electrons and

holes are formed. The electrons have been promoted to the conduction band
then migrates toward the metallic film. The liberated electrons are free to
migrate through the external circuit. As a result an electrical current of a
magnitude is proportional to the number of photons that strike the
semiconductor surface.
Advantages
Rugged, low cost, No external source of electrical energy required.
Disadvantages
Low internal resistance of the cell makes the amplification of its output. Less
convenient, Suffers from lack of sensitivity at low levels.
Vacuum phototubes
A photo-emissive detector inside an evacuated envelope with a transparent

window is the photocathode. The photocathode can be opaque or
semitransparent. The anode is a thin wire inside the photocathode.

The concave surface of the electrode supports a layer of photo emissive

material that tends to emit electrons when it is irradiated when a voltage is
applied. The emitted electron flow to the wire anode generating a photocurrent
that is generally about one tenth as great as that associated with photovoltaic
cell. The number of electrons ejected from a photo emissive surface is directly
proportional to the radiant power or beam that strikes the surface.
Photomultiplier tube
Photomultiplier tubes are limited to measuring low power radiation because

intense light causes irreversible damage to photomultiplier tube. For the
measurement of low radiant powers, the photomultiplier tube offers
advantages over an ordinary phototubes and it emits electrons when exposed
to radiation. The tube also contains additional electrodes called Dynodes.
Dynode D1 is maintained at a voltage 90V more positive than cathode and

electrons are accelerated towards it. Each photoelectron that strikes the
dynode causes emission of several additional electrons. These electrons in
turn are accelerated toward dynode D2 which 90V more positive than dynode
D1, again several electrons are emitted for each electron that strikes the
surface. By the times this process is repeated several times 106 to 107 electrons
get formed for each incident photon.

Thus cascade of electrons is finally collected at the anode and the resulting
current is then converted to voltage and measured.
Disadvantages
Photomultiplier tubes are limited to measuring low power radiation because

intense light causes irreversible damage to photomultiplier tube.
E) Signal processors and readout system
The signal processor is usually an electronic device that amplifies the electric
signal from the transducer. It may alter the signal from DC (Direct current) to
AC (Alternating current), change the phase of the signal and filter it to remove
unwanted components. They may perform mathematical operations on the
signals as differentiation, integration, or conversion to logarithm. Several
types of readout devices are found in modern instruments. Some include
digital meters, recorders, cathode ray tubes, LCD panels and computer
displays.
INSTRUMENTS
Single Beam Spectrophotometer
It consist of a tungsten or deuterium lamp, a filter or a monochromator for

wavelength selection. Matched cells then can be placed alternately in radiation
beam.

Normally a single beam instrument requires a stabilized voltage supply to

avoid error resulting from changes in the beam intensity during the time
required to make the hundred percent measurement and determine percent
for the analyte.
The simplest and least expensive one consist of a battery operated tungsten
bulb as a source, a set of glass filters for wavelength selection, test-tube or
sample holder , a transducer and read out device.
Double Beam Spectrophotometer
In this type of instrument, the monochromatic light is split by a rapidly

rotating beam chopper into two beams which are directed alternately in rapid
succession through a cell containing sample and one containing the solvent
only. If there is great absorption of light in the sample cell than in the reference
cell, the recombination of the beams at the detector produces a pulsating
current which is converted into two direct current voltages proportional to the
light intensities.
Recording spectrophotometers are double beam instruments equipped with a

wavelength scanning device which allow the rapid automatic scanning of
spectra.

Applications of UV Visible spectrophotometry
1. Detection of Impurities
UV absorption spectroscopy is one of the best methods for determination of
impurities in organic molecules. Additional peaks can be observed due to
impurities in the sample and it can be compared with that of standard raw
material. By also measuring the absorbance at specific wavelength, the impurities
can be detected. Benzene appears as a common impurity in cyclohexane. Its
presence can be easily detected by its absorption at 255 nm.
2. Structure elucidation of organic compounds.
UV spectroscopy is useful in the structure elucidation of organic molecules, to
identify the presence or absence of unsaturation, the presence of hetero atoms.
From the location of peaks and combination of peaks, it can be concluded that
whether the compound is saturated or unsaturated, hetero atoms are present or
not etc.
3. Quantitative analysis
UV absorption spectroscopy can be used for the quantitative determination of
compounds that absorb UV radiation. This determination is based on Beer’s law.
4. Qualitative analysis
UV absorption spectroscopy can characterize those types of compounds which
absorbs UV radiation. Identification is done by comparing the absorption
spectrum with the spectra of known compounds.

UV absorption spectroscopy is generally used for characterizing aromatic

compounds and aromatic olefins.
Result: The principle, instrumentation and applications of UV visible

spectrophotometry were studied.
Questions for Viva and/ synopsis

 How can we perform qualitative analysis using UV visible
spectrophotometry?
 Which are the different radiation sources used in UV Visible
spectrophotometer?
 How does grating monochromator work?
 Why the cuvettes are made of Quartz?
 Why baseline correction is needed in double beam UV Visible
spectrophotometer?
 What is Beer Lambert’s law? Give its importance

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EXERCISE NO 2 Date:
AIM: To study Jasco UV Visible spectrophotometer V-730.
Reference: Jasco UV Visible spectrophotometer V-730 Manual
Instrument: UV Visible Spectrophotometer
Make: Jasco
Model: V-730
Software: Spectra Manager
Specifications:
Optical System Rowland off-circle arrangement
Single monochromator
Double beam type

Light source Halogen lamp, Deuterium lamp
Wavelength range 190 to 1100 nm
Wavelength +/-0.2 nm (at 656.1 nm)
accuracy
Wavelength +/-0.1 nm
repeatability
Spectral 1 nm
bandwidth (SBW)
Scanning speed 10-8000 nm/min
Slew speed 24,000 nm/min
Detector Silicon photodiode
Standard IQ accessories, Start button, Analog output
functions
Standard Abs/%T meter, Quantitative analysis, Spectrum
programs measurement,
Time course measurement, Fixed wavelength
measurement, Validation, Daily maintenance, Two
wavelength time course measurement
Dimensions and 486(W) x 441(D) x 216(H) mm, 15 kg
weight
Installation Room temperature: 15-30 Celsius, humidity: below 85%
requirements
Software Spectra Manager

Operating procedure
1. Switch ON mains Power supply.

2. Switch ON the power button on right side of the instrument.
3. The START button will start blinking with blue light. Wait till the
blinking stops. This indicates that the Initialization process is over.
4. Switch on the PC and double click on Spectra Manager icon on the
desktop. Then click on Spectrum Measurement.
5. Go to Measure and set the parameters as per the requirement.
6. Place the solvent in both cuvettes and click on B (For Baseline
Correction).
7. Place the sample in front cuvette and click on S ( For sample
measurement)
8. The completion of measurement of absorbance spectra opens a spectra
analysis window.
9. This window provides different tools for analysis of spectra.
10. Use the tools as per requirement.
Shut down procedure
1. Verify that the sample compartment is empty.

2. Close the spectra manager window, Spectra Measurement Window and
Spectra Analysis Window.
3. Switch OFF the power button of instrument and Shut down the
computer.
Result: The features of Jasco UV Visible spectrophotometer were studied.

EXPERIMENT NO 1 Date:
AIM: To perform calibration of UV Visible spectrophotometer.
Reference
Pharmacopoeia, I., 2014. Govt. of India, Ministry of Health and Family

Welfare, Delhi, Ghaziabad, Vol. II, A76 – A77
Requirements
Chemicals: Potassium Dichromate, Sulphuric acid, Potassium Chloride,

Toulene, Hexane, Distilled Water.
Glassware: Volumetric flask 100ml, volumetric flask 10ml, Pipette, butter

paper, spatula
Instrument: UV Visible spectrophotometer JASCO Model: V-730
Theory
Importance of calibration
Calibration is a comparison between a known measurement (the standard)

and the measurement using your instrument. Typically, the accuracy of the
standard should be ten times the accuracy of the measuring device being
tested. However, accuracy ratio of 3:1 is acceptable by most standards
organizations.
Calibration of instruments has two objectives. It checks the accuracy of the

instrument and it determines the traceability of the measurement. In practice,
calibration also includes repair of the device if it is out of calibration. A report
is provided by the calibration expert, which shows the error in measurements
with the measuring device before and after the calibration.
The accuracy of all measuring devices degrade over time. This is typically
caused by normal wear and tear. However, changes in accuracy can also be
caused by electric or mechanical shock or a hazardous manufacturing
environment (ex- oils, metal chips etc.).

Depending on the type of the instrument and the environment in which it is

being used, it may degrade very quickly or over a long period of time. The
bottom line is that, calibration improves the accuracy of the measuring device.
Accurate measuring devices improve product quality.
Calibration is required for -
 Testing a new instrument
 Testing an instrument after it has been repaired or modified
 Periodic testing of instruments
 Testing after the specific usage has elapsed
 Prior to and/or after a critical measurement
 When observations are not accurate or instrument indicators do not

match the output of a surrogate instrument
 After events such as- An instrument has had a shock, vibration, or

exposure to adverse conditions, which can put it out of calibration or
damage it.
 Sudden weather changes
Procedure
In order to calibrate the UV Visible spectrophotometer, following parameters

are recommended-
1. Control of wavelength
2. Control of absorbance
3. Limit of stray light
4. Resolution power
5. Spectral slit width
In this experiment, the UV visible spectrophotometer is calibrated for control

of absorbance, limit of stray light and resolution power.

Control of Absorbance
For Ultra violet region:
1. Dissolve 0.027 ml of sulphuric acid in 100ml distilled water. (0.005

M Sulphuric acid).
2. Weigh accurately 57.0 to 63.0 mg of Potassium dichromate

primary standard (previously dried at 130°C for constant weight)
and transfer it to 1000 ml volumetric flask. Dissolve in 0.005M
sulphuric acid and make up to the mark with the same acid.
 Measure the absorbance at 235nm, 257nm, 313nm and 350nm

using 0.005M sulphuric acid as reference solution.
For visible region:
1. Weigh accurately 57.0 to 63.0 mg of Potassium dichromate

primary Standard (previously dried at 130°C for constant weight) and
transfer it to 100 ml volumetric flask.
2. Dissolve in 0.005M sulphuric acid and make up to the mark with the
same acid.
 Measure the absorbance at 430nm using sulphuric acid as

reference solution.
 Calculate the value of A(1% , 1 cm ) using the expression given below :
Absorbance
A1%
1cm =
Weight in gm per 100ml

 Check the value of A (1%, 1 cm ) at each wavelength against the

acceptance criteria given below :
Wavelength Maximum Tolerance
235 nm 122.9 to 126.2
257 nm 142.4 to 145.7
313 nm 47.0 to 50.3
350 nm 104.9 to 108.2
430 nm 15.7 to 16.1
Limit of stray light
 Prepare a solution of 1.2 % w/v of Potassium chloride (previously dried

at 105°C, 2 hours) in distilled water.
 Determine the absorbance using path length of 1 cm at 200 nm against

purified water as blank.
 Acceptance criteria : Absorbance at 200 nm should be greater than

2.0
Resolution power
 Prepare a solution of 0.02 % v/v Toluene in Hexane as follows:
 Dilute 2.0 ml of toluene to 100.0 ml with hexane. Further dilute 1.0 ml

this solution to 100.0 ml with hexane.
 Record the spectrum of 0.02% v/v solution of toluene in hexane from

wavelength 250nm to the wavelength 300 nm.
 Record the absorbance at the maxima at about 269 nm and minima at

about 266
 Calculate the ratio of absorbance by dividing the absorbance at the

maxima at about 269nm and minima at about 266nm

 Acceptance criteria: The ratio of Absorbance at the maxima at

about 269 nm to that at the minimum at about 266 nm is not less
than 1.5.
Observation
Control of Absorbance
Wavelength Absorbance 𝐀𝟏%

𝟏𝐜𝐦 Maximum Tolerance
235 nm 122.9 to 126.2
257 nm 142.8 to 146.2
313 nm 47.0 to 50.3
350 nm 105.6 to 109.0
430 nm 15.7 to 16.1
Limit of stray light
Absorbance at 200nm was found to be-
Resolution power
Absorbance at 269nm =
Absorbance at 266 nm =
Ratio of Absorbance at the maxima at about 269 nm to that at the minimum

at about 266 nm =
Result
1. Control of absorbance: As the calculated values for A1%

1cm solution of
potassium dichromate UV at the recommended wavelengths fall / do not fall

in the prescribed range of maximum tolerance, the instrument is/ is not
calibrated for control of absorbance.

2. Limit of stray light: The absorbance of 1.2% w/v solution of KCl , at

200 nm was found to be . This value is greater than/ less than 2.0.
Hence, the instrument is / is not calibrated for limit of stray light.
3. Resolution power: The ratio of absorbance of 0.02%v/v Toulene in

hexane solution at 269nm to that at 266 nm was found to be . This
value is greater than/ less than 1.5. Hence the instrument is / is not
calibrated for resolution power.

 What is calibration of instrument? Give its importance.
 What is stray light?
 What is resolution?
 How do you calibrate UV Visible spectrophotometer? Enlist all the
parameters.

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EXERCISE NO 3 Date:
AIM: To study Multicomponent analysis by UV Visible

spectrophotometer.
Reference
1. Beckett, A.H. and Stenlake, J.B. eds., 1988. Practical Pharmaceutical

Chemistry: Part II Fourth Edition (Vol. 2). A&C Black.
2. Kamal, A.H., El-Malla, S.F. and Hammad, S.F., 2016. A review on UV

spectrophotometric methods for simultaneous multicomponent analysis. Eur
J Pharm Med Res, 3(2), page 348-60.
Theory
Combination drug products occupy a time-honored and important role in

therapeutics. When rationally formulated, fixed-combination drugs may
produce greater convenience, lower cost, and sometimes greater efficacy and
safety. Analysis of samples with numerous components presents a major
challenge in modern analysis. Multi-component analysis has become one of
the most appealing topics for analytical chemists in the last few years, in fields
as clinical chemistry, drug analysis, pollution control etc.
Different analytical techniques can be applied for multicomponent analysis

including; spectrophotometry, chromatography, and electrophoresis. The use
of traditional methods like extraction is quite difficult because extraction
techniques require large solvent consumption, with accompanying risks of
analyte loss or contamination, and possibility of incomplete separation. The
procedure may be expensive and time consuming.
UV spectrophotometric techniques are mainly used for multicomponent

analysis thus minimizing the cumbersome task of separating interferents and
allowing the determination of an increasing number of analytes, consequently
reducing analysis time and cost.

Multicomponent UV spectrophotometric methods are based on recording and

mathematically processing absorption spectra. They offer the following
advantages:
 Avoiding prior separation techniques e.g. extraction, concentration of

constituents, and cleanup steps that might be required
 Spectral data are readily acquired with ease
 The process is fast, accurate, and simple
 Wide applicability to both organic and inorganic systems
 Typical detection limits of 10-4 to 10-5 M and moderate to high selectivity.
Different UV spectrophotometric multicomponent analysis methods include-
1-Simultaneous equation method
If a sample contains two absorbing drugs (x and y) each of which absorbs at

the λmax of the other, it may be possible to determine both drugs by the
technique of simultaneous equation (Vierordt’s method) provided that certain
criteria apply-
1. The λmax of both the drugs in combination should be far apart from
each other.
2. Both the drugs should show some absorbance on each other’s λmax.
3. Both the drugs should have solubility in same solvent system.
4. Both the drugs should not interact with each other.
The information required is-
 The absorptivities of x at λ1 and λ2, ax1and ax2 respectively

 The absorptivities of y at λ1 and λ2, ay1and ay2respectively
 The absorbance of the mixture samples at λ1and λ2, A1 and A2
respectively.
As Beer Lambert’s law says- A= abc
Where, A = Absorbance
a = Absorptivity

b = Path length (1cm)
C = Concentration
Let Cx and Cy be the concentration of x and y respectively in the mixture

samples. Two equations are constructed based upon the fact that at λ1 and at
λ2, the absorbance of the mixture is the sum of the individual absorbance of
x and y -
At λ1, A1 = ax1 b Cx (Absorbance of drug X) + ay1 b Cy (Absorbance of drug Y )…..I
At λ2, A1 = ax2 b Cx (Absorbance of drug X) + ay2 b Cy (Absorbance of drug Y )…..II
In equation I and II, the absorbance is written in expanded form as per Beer
Lambert’s law. Solving the equations I and II for CX and CY gives concentrations
of drug X and Y.
Alternatively rearrangement of equation II give –
A2 − ax2 b Cx
Cy = ….. III
ay2
Substituting this value of Cy In equation I and II and rearranging gives-
A2 ay1 − A1 ay2
Cx = ….IV
ax2 ay1 − ax1 ay2
A1 ax2 − A2 ax1
Cy = ….V
ax2 ay1 − ax1 ay2
Solving the equations IV and V gives concentrations of drug X and Y.
Criteria for obtaining maximum precision based upon absorbance ratios have
been suggested. The criteria are that the ratios-
𝐴2 / 𝐴1 𝐴2 / 𝐴1
and
𝑎𝑥2 / 𝑎𝑥1 𝑎𝑦2 / 𝑎𝑦1
should lie outside the range 0.1‐ 2.0 for the precise determination of X and Y
respectively. These criteria are satisfied only when the λmax of two component
are reasonably dissimilar.

2- Q –Method/ Iso-absorptive point method
This method "also termed absorption ratio method" is a modification of the

simultaneous equations method. According to this method, the ratio of
absorbance at any two wavelengths for a substance, which obeys Beer's law,
is a constant value independent of the concentration and path length. This
constant is termed as "Hufner's Quotient" or Q-value. The method involves the
measurement of absorbance at two wavelengths, one being the λmax of one of
the components (λ2) and the other being a wavelength of equal absorptivity of
the two components (λ1), called the iso-absorptive/ iso-bestic / Q point.
Q-point /Iso-absorptive point is a wavelength which is equally absorbed by

both the drugs in a mixture. The corresponding wavelength is considered as
λ1. The λmax of either drug X or drug Y is considered as λ2. Usually the λmax
which is more distant to Q point wavelength is the choice.
The concentration of each component can be calculated by mathematical

equations –
Qm − Qy A1
CX = ×
QX − QY ax1

Qm − Qx A1
CY = ×
Qy − Qx ay1
Where,
CX = Concentration of drug X
CY = Concentration of drug Y
A1 = Absorbance of mixture sample at λ1 (Iso-absorptive wavelength)
ax1 = Absorptivity of drug X at λ1
ay1 = Absorptivity of drug Y at λ1
Absorbance of sample mixture at λ1

QM =
Absorbance of sample mixture at λ2
Absorbptivity of drug X at λ1
QX =
Absorbptivity of drug X at λ2
Absorbptivity of drug Y at λ1
QY =
Absorbptivity of drug Y at λ2
Using these equations the concentrations of drug X and drug Y can be

calculated.
 What is multicomponent analysis?

 Enlist different methods for multicomponent analysis by UV Visible
spectrophotometry.
 Explain in brief principle of Simultaneous equation method.
 Explain in brief principle of Q-Analysis method.
 Give pre-requisites for application of simultaneous equation method.
 What is iso-absorptive point?

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AIM: To perform Simultaneous equation method for estimation

of Paracetamol and Aceclofenac in bulk mixture.
Reference:

Chemistry: Part II Fourth Edition (Vol. 2). A&C Black, page -275.
2. Mahaparale, P.R., Sangshetti, J.N. and Kuchekar, B.S., 2007.
Simultaneous spectrophotometric estimation of Aceclofenac and
Paracetamol in tablet dosage form, Indian Journal of Pharmaceutical
Sciences, 69(2), p.289.
3. Pharmacopeia, I., 2007. Vol-I and II Indian Pharmacopeia Commission.
Ghaziabad, Govt. of India: Ministry of Health and Family Welfare, p.514-
571.
Requirements
1. Apparatus: Glass beakers, volumetric flask, funnel, graduated pipette,

etc.
2. Chemicals: Paracetamol, Aceclofenac, Methanol (AR Grade) etc.
3. Instrument: UV Visible Spectrophotometer, Sonicator.
Theory
Refer exercise no 3 for theory of simultaneous equation method.
Paracetamol – Drug profile

1) Name- Paracetamol
3) Molecular Formula- C8H9NO2
4) Molecular weight- 151.2
5) Description- White crystal powder or White crystalline powder.
6) Action- Analgesic, Anti-inflammatory.
7) Uses- Anti-pyretic.
8) Marketed preparation- Crocin, Paracin, Calpol.
Aceclofenac – Drug profile
1) Name- Aceclofenac
3) Molecular Formula- C16H13Cl2NO4
6) Action- Analgesic, Anti-inflammatory, Anti-pyretic.
7) Uses- Rheumatoid arthritis osteoarthritis.
8) Marketed preparation- Fico, Acemove, Nopenac.
Procedure
Preparation of stock solution
Weigh accurately about 10 mg of Paracetamol and Aceclofenac separately and

dissolve in 100 ml of Methanol to get the stock solution of 100 µg/ml.

Preparation of standard solutions of Paracetamol
Pipette out ml, ml, ml, ml and ml from the stock solution of
Paracetamol in separate 10ml volumetric flasks and dilute each up to 10 ml
with distilled water to get µg/ml, µg/ml, µg/ml, µg/ml and µg/ml
concentrations of standard solutions of Paracetamol.
Preparation of standard solutions of Aceclofenac
Aceclofenac in separate 10ml volumetric flasks and dilute each up to 10 ml
concentrations of standard solutions of Aceclofenac.
Preparation of bulk mixture
Pipette out 1 ml from Paracetamol stock solution and 1 ml from Aceclofenac

stock solution, mix and dilute up to 10 ml with distilled water. Prepare such
5 bulk mixtures.
Observations
λmax of Paracetamol = nm = λ1
λmax of Aceclofenac = nm = λ2

Absorbance of standard solutions of Paracetamol (Drug X) and calculation

of absorptivities
Concentration Absorbance Absorptivity Absorbance Absorptivity

(µg/ml) at λ1 (ax1) at λ2 (ax2)
Average Average
ax1 = ax2 =
Absorbance of standard solutions of Aceclofenac (Drug Y) and calculation

of absorptivities
Concentration Absorbance Absorbance

Absorptivity Absorptivity
(µg/ml) at λ1 at λ2
Average Average
ay1 = ay2 =

Absorbance of bulk mixture
Mixture Absorbance at λ1 Absorbance at λ2

No. A1 A2
Calculations

Result
Theoretical Concentration
Concentration found (Avg) Standard %
DRUG
Deviation Found
(µg/ml) (µg/ml)
Paracetamol (X)
Aceclofenac (Y)
 What is simultaneous equation method?

 Give category of Paracetamol and Aceclofenac.

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of Paracetamol and Aceclofenac in tablet mixture.
Reference:

Sciences, 69(2), p.289.
571.
Requirements

etc.
Theory

Aceclofenac –Drug profile
Procedure


Preparation of tablet mixture
Calculate the average weight of two tablets. Calculate the equivalent weight of
tablet containing 10 mg of Aceclofenac and 32.5 mg of Paracetamol. Dissolve
the equivalent weight in 100 ml of Methanol. Sonicate for 10 min and filter
through the Whatmann filter. This gives a tablet stock solution containing 325
µg/ml of Aceclofenac and 100 µg/ml of Paracetamol. Pipette out 0.5 ml of
tablet stock solution and dilute up to 10 ml with distilled water. Prepare such
5 samples.
Observations:


of absorptivities

Average Average
ax1 = ax2 =

of absorptivities

Average Average
ay1 = ay2 =

Absorbance of Tablet mixture

No. A1 A2
Calculations

Result
Standard %
DRUG Concentration found (Avg)
Deviation Assay
(µg/ml) (µg/ml)
Paracetamol (X)
Aceclofenac (Y)

 Give category of Paracetamol and Aceclofenac.

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of Paracetamol and Ibuprofen in Bulk mixture.
Reference:

Sciences, 69(2), p.289.
571.
Requirements

etc.
2. Chemicals: Paracetamol, Ibuprofen, Methanol (AR Grade) etc.
Theory

Ibuprofen – Drug Profile
1) Name-Ibuprofen
3) Molecular Formula- C13H18O2
4) Molecular weight- 206.28 g/mol
5) IUPAC name: 2-[4-(2-methylpropyl) phenyl] propionic acid
6) Action- non-steroidal anti-inflammatory drug
7) Uses- Treatment of pain or inflammation caused by many conditions such

as headache, toothache, back pain, arthritis, menstrual cramps, or minor
injury.
8) Marketed preparation- On the available products, ibuprofen is administered

as a racemic mixture. Once administered, the R-enantiomer undergoes
extensive interconversion to the S-enantiomer in vivo by the activity of the
alpha-methylacyl-CoA racemase. In particular, it is generally proposed that
the S-enantiomer is capable of eliciting stronger pharmacological activity than
the R-enantiomer

Procedure
Weigh accurately about 10 mg of Paracetamol and Ibuprofen separately and

Preparation of standard solutions of Ibuprofen
Ibuprofen in separate 10ml volumetric flasks and dilute each up to 10 ml with
distilled water to get µg/ml, µg/ml, µg/ml, µg/ml and µg/ml
concentrations of standard solutions of Ibuprofen.
Pipette out 1 ml from Paracetamol stock solution and 1 ml from Ibuprofen

5 bulk mixtures.
Observations:
λmax of Ibuprofen = nm = λ2


of absorptivities

Average Average
ax1 = ax2 =
Absorbance of standard solutions of Ibuprofen (Drug Y) and calculation of

absorptivities

Average Average
ay1 = ay2 =

Absorbance of Bulk mixture

No. A1 A2
Calculations

Result
DRUG
Deviation Found
(µg/ml) (µg/ml)
Paracetamol (X)
Ibuprofen (Y)

 Give category of Paracetamol and Ibuprofen.

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of Paracetamol and Ibuprofen in Tablet mixture.
Reference

Sciences, 69(2), p.289.
571.
Requirements

etc.
Theory

Ibuprofen –Drug profile
1) Name- Ibuprofen

injury.

alpha-methylacyl-CoA racemase.

In particular, it is generally proposed that the S-enantiomer is capable of

eliciting stronger pharmacological activity than the R-enantiomer
Procedure

tablet containing 10 mg of Ibuprofen and 8.125 mg of Paracetamol. Dissolve
in 100 ml of Methanol. Sonicate for 10 min and filter through the Whatmann
filter. This gives a tablet stock solution containing 100 µg/ml of Ibuprofen and
8 µg/ml of Paracetamol.
Pipette out ml of tablet stock solution and dilute up to 10 ml with distilled

water. Prepare such 5 samples.

Observations:

of absorptivities

Average Average
ax1 = ax2 =

absorptivities

Average Average
ay1 = ay2 =


No. A1 A2
Calculations

Result
Standard %
Deviation Assay
(µg/ml) (µg/ml)
Paracetamol (X)
Ibuprofen (Y)

 Give category of Paracetamol and Ibuprofen.

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of Paracetamol and Tramadol in Bulk mixture.
Reference:

2. El-Zinati, A.M. and Abdel-Latif, M.S., 2015. Simultaneous determination
of paracetamol and tramadol in pharmaceutical tablets by derivative UV-
Vis absorption spectrophotometry. Simultaneous determination of
paracetamol and tramadol in pharmaceutical tablets by derivative UV-Vis
absorption spectrophotometry, 8(1).
571.
Requirements:

etc.
2. Chemicals: Paracetamol, Tramadol, Methanol (AR Grade) etc.
Theory

Tramadol – Drug profile
1) Name- Tramadol
5) IUPAC name: (1R,2R)-2-[(dimethylamino)methyl]-1-(3- methoxyphenyl)

cyclohexan-1-ol
6) Action- Tramadol is a synthetic codeine analogue, Tramadol has central

analgesic properties with effects similar to opioids, such as morphine and
codeine, acting on specific opioid receptors. Used as a narcotic analgesic for
severe pain, it can be addictive and weakly inhibits norepinephrine and
serotonin reuptake
7) Uses- Used as a narcotic analgesic for severe pain, it can be addictive and
weakly inhibits norepinephrine and serotonin reuptake

Procedure
Weigh accurately about 10 mg of Paracetamol and Tramadol separately and

dissolve in 10 ml of Ethanol. Make up the volume up to 100ml with distilled
water to get the stock solutions of 100 µg/ml.
Paracetamol in separate 10ml volumetric flasks and dissolve in 1 ml of
Ethanol. Dilute each up to 10 ml with distilled water to get µg/ml, µg/ml,
µg/ml, µg/ml and µg/ml concentrations of standard solutions of
Paracetamol.
Preparation of standard solutions of Tramadol
Pipette out ml, ml, ml, ml and ml from the stock solution of Tramadol
in separate 10ml volumetric flasks and dissolve in 1 ml of Ethanol. Dilute
each up to 10 ml with distilled water to get µg/ml, µg/ml, µg/ml, µg/ml
and µg/ml concentrations of standard solutions of Tramadol.
Pipette out 1 ml from Paracetamol stock solution and 1 ml from Tramadol

5 bulk mixtures.
Observations:
λmax of Tramadol = nm = λ2

of absorptivities


Average Average
ax1 = ax2 =
Absorbance of standard solutions of Tramadol (Drug Y) and calculation of

absorptivities

Average Average
ay1 = ay2 =


No. A1 A2
Calculations

Result
DRUG
Deviation Found
(µg/ml) (µg/ml)
Paracetamol (X)
Tramadol (Y)

 Give category of Paracetamol and Tramadol.

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of Paracetamol and Tramadol in Tablet mixture.
Reference

2. El-Zinati, A.M. and Abdel-Latif, M.S., 2015. Simultaneous determination
of Paracetamol and tramadol in pharmaceutical tablets by derivative UV-
Vis absorption spectrophotometry. Simultaneous determination of
Paracetamol and tramadol in pharmaceutical tablets by derivative UV-Vis
absorption spectrophotometry, 8(1).
571.
Requirements

etc.
2. Chemicals: Paracetamol, Tramadol, Methanol (AR Grade) etc.
Theory

Tramadol
1) Name- Tramadol
5) IUPAC name: (1R,2R)-2-[(dimethylamino)methyl]-1-(3-

methoxyphenyl)cyclohexan-1-ol
6) Action- Tramadol is a synthetic codeine analogue, Tramadol has central

analgesic properties with effects similar to opioids, such as morphine and
codeine, acting on specific opioid receptors. Used as a narcotic analgesic for
severe pain, it can be addictive and weakly inhibits norepinephrine and
serotonin reuptake
7) Uses- Used as a narcotic analgesic for severe pain, it can be addictive and
weakly inhibits norepinephrine and serotonin reuptake

Procedure
Weigh accurately about 10 mg of Paracetamol and Tramadol separately and

dissolve in 10 ml of Ethanol. Make up the volume up to 100ml with distilled
water to get the stock solutions of 100 µg/ml.
Paracetamol in separate 10ml volumetric flasks and dissolve in 1 ml of
Ethanol. Dilute each up to 10 ml with distilled water to get µg/ml, µg/ml,
µg/ml, µg/ml and µg/ml concentrations of standard solutions of
Paracetamol.
Preparation of standard solutions of Tramadol
Pipette out ml, ml, ml, ml and ml from the stock solution of Tramadol
in separate 10ml volumetric flasks and dissolve in 1 ml of Ethanol. Dilute
each up to 10 ml with distilled water to get µg/ml, µg/ml, µg/ml, µg/ml
and µg/ml concentrations of standard solutions of Tramadol.
Preparation of Tablet mixture
tablet containing mg of Paracetamol and mg of Tramadol. Dissolve in
10 ml of Ethanol. Dilute up to 100 ml with distilled water. Sonicate for 10 min
and filter through the Whatmann filter. This gives a tablet stock solution
containing µg/ml of Paracetamol and µg/ml of Tramadol.
Pipette out ml of tablet stock solution and dilute up to 10 ml with 1 ml

of Ethanol and 9 ml of distilled water. Prepare such 5 samples.
Observations:
λmax of Tramadol = nm = λ2


of absorptivities
Concentratio Absorptivit
Absorbance Absorptivity Absorbanc
n y
at λ1 (ax1) e at λ2
(µg/ml) (ax2)
Average Average
ax1 = ax2 =
Absorbance of standard solutions of Tramadol (Drug Y) and calculation of

absorptivities

Average Average
ay1 = ay2 =


No. A1 A2
Calculations

Result
Standard %
Deviation Assay
(µg/ml) (µg/ml)
Paracetamol (X)
Tramadol (Y)

 Give category of Paracetamol and Tramadol.

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EXERCISE NO 4 Date:
AIM: To study principle, instrumentation and applications of

Infrared Spectroscopy.
Reference:
1. Skoog, D.A., Holler, F.J. and Crouch, S.R., 2017. Principles of instrumental
analysis. Cengage learning.page-256-320.
2. Willard, H.H., Merrit, L.L., Dean, J.A. and Settle, F.A., Instrumental method
of analysis, 7th edition, CBS Publisher and Distributors, page.417-428.
3. Sharma, Y.R., 2009. Elementary organic spectroscopy, principles and

chemical application. Chand and Company Ltd, New Delhi, India, page.137-
138.
Theory
Spectroscopy can be defined as the interaction between matter and radiation.

Infrared spectroscopy is a very powerful technique which uses
electromagnetic radiation in the infrared region for the determination and
identification of molecular structure as well as having various
quantitative applications within analytical chemistry. It is important to note
that atoms can absorb energy from electromagnetic radiation, this absorbed
energy alters the state of the atoms within the molecule. These changes are
usually manifest in alterations to the frequency and amplitude of
molecular vibrations, which may be measured and plotted to produce an
infrared spectrum. The term "infra-red" covers the range of the
electromagnetic spectrum wavelengths between 0.78 and 1000 µm. In the
context of infra-red spectroscopy, wavelength is measured in "wavenumbers",
with the unit cm-1.
1
WAVENUMBER =
WAVELENGTH IN CENTIMETER

Infrared spectrometers use optical devices for dispersing and focusing

electromagnetic radiation of IR frequency which is passed through the sample
and any changes in absorbance measured against a reference beam. There are
three well defined IR regions (near, mid and far). The boundaries between them
are not clearly defined, but broadly they are defined as
Region Wavelength range (µm) Wavenumber range (cm-1)
Near 0.78 - 2.5 12800 - 4000
Middle 2.5 - 50 4000 - 200
Far 50 -1000 200 - 10
The low energies, typically encountered within the infrared region, are
not sufficient to cause electronic transitions; however, they are large
enough to cause changes in the frequency and amplitude of molecular
vibrations. IR Spectroscopy detects frequencies of infrared light that are
absorbed by a molecule. Molecules tend to absorb these specific frequencies
of light since they correspond to the frequency of the vibration of bonds in the
molecule.
The energy required to excite the bonds belonging to a molecule, and to make
them vibrate with more amplitude, occurs in the Infrared region. A bond will
only interact with the electromagnetic infrared radiation, however, if it is polar.
The presence of separate areas of partial positive and negative charge in a
molecule allows the electric field component of the electromagnetic wave to
excite the vibrational energy of the molecule. The change in the vibrational
energy leads to another corresponding change in the dipole moment of the
given molecule. The intensity of the absorption depends on the polarity of the
bond. Symmetrical non-polar bonds in N≡N and O=O do not absorb radiation,
as they cannot interact with an electric field.

Most of the bands that indicate what functional group is present are found in
the region from 4000 cm-1 to 1300 cm-1. Their bands can be identified and
used to determine the functional group of an unknown compound. Bands that
are unique to each molecule, similar to a fingerprint, are found in the
fingerprint region, from 1300 cm-1 to 400 cm-1. These bands are only used to
compare the spectra of one compound to another.
A molecule can be identified by comparing its absorption peak to a data

bank of spectra. IR spectroscopy is very useful in the identification and
structure analysis of a variety of substances, including both organic and
inorganic compounds. It can also be used for both qualitative and quantitative
analysis of complex mixtures of similar compounds.
PRINCIPLE
The study of principle of IR includes study of following concepts-
1. Vibrational motion.
2. Change in dipole moment.
3. Fundamental vibration.
4. Symmetry and IR spectra.
5. Overtone peak and Combination peak.
6. Coupled vibration.
7. Fermi resonance.
Vibrational motion
All the bonds within a molecule undergo vibrations. These vibrations result in
a specific lowest vibrational energy for the molecule. Each bond which
undergoes vibrations generates an electromagnetic field surrounding the bond
which has specific frequency. If the provided frequency matches with the
generated frequency of that bond and it results in change in dipole moment,
then the provided frequency is absorbed by that bond. After the absorption of

specific frequency, the bond vibrates with a higher amplitude. The resultant
transmittance of frequency is recorded to give IR spectra. A plot of frequencies
on X axis and % transmittance on Y-axis gives IR spectra.
Change in dipole moment
To absorb IR radiation, a molecule must undergo a net change in dipole

moment as it vibrates or rotates. Only under these circumstances, the
alternating electric field of radiation can interact with the molecule and cause
changes in the amplitude of its vibration. In general, molecular vibrations
which will lead to a change in dipole moment of the molecule will give rise to
absorption bands in the infrared region. Otherwise they are said to be inactive
and will show no absorption.
The dipole moment is determined by the magnitude of charge difference and

the distance between two centers of charge. A large change in dipole moment
will usually give rise to strong absorption. For example, the symmetrical
stretching of C=C bond will not produce a change in dipole moment of the
molecule. Therefore, this mode of vibration is infrared inactive.
Fundamental vibrations

Vibration fall into basic categories of stretching and bending. Stretching

vibrations result in change in the bond length of the bond while bending
vibrations involve change in the angle between two bands.
The stretching and bending vibrations are classified as follows-
The symbols ‘+’ and ‘-’ indicate above the plane and below the plane
vibrations.
Scissoring-Both atom move towards each other.
Rocking-Both moving in same direction.
Wagging-Both move together out of plane.
Twisting-both move out of plane but in opposite direction.
The number of fundamental vibrations and thus theoretical number of peaks

in Infrared spectra of a known compound can be calculated if the number of
atoms within the molecule is known. This calculation is based on the following
equations-
3n-6 for a non-linear molecule and 3n-5 for a linear molecule, where n
indicates the number of atoms in the molecule.

Symmetry and IR spectra
The molecules with more symmetric centers usually reduce the number of
peaks in IR spectra.
Overtone peak and combination peak
In vibrational spectroscopy, an overtone band is the spectral band that occurs

in a vibrational spectrum of a molecule when the molecule makes a transition
from the ground state (v=0) to the second excited state (v=2), where v is the
vibrational energy level. A combination peak is observed when a single photon
has precisely the correct energy to excite two vibrations at once. The energy of
the combination band must be the exact sum of the two independent
absorptions.
Coupled Vibration
Vibrational coupling takes place between two bonds through a common atom.
If the bonds are reasonably close in a molecule, the coupling vibrations may
be both fundamental vibrations and a fundamental vibration coupled with
overtone of some other vibration. Example – Carbon di-oxide molecule has two
C=O bonds with a common carbon atom. It has two fundamental stretching
vibrations and coupled vibration is seen at frequency 2350cm-1. Whereas, the
absorption for the carbonyl group in an aliphatic ketone is observed around
1715cm-1.
Fermi resonance
Combination of overtone peak of a bond and fundamental peak of particular

bond results in combination called Fermi Resonance.

INSTRUMENTATION
An infrared spectrophotometer is an instrument that passes infrared light

through an organic molecule and produces a spectrum that contains a plot of
the amount of light transmitted on the vertical axis against the wavelength of
infrared radiation on the horizontal axis. In infrared spectra the absorption
peaks point downward because the vertical axis is the percentage
transmittance of the radiation through the sample. Absorption of radiation
lowers the percentage transmittance value. Since all bonds in an organic
molecule interact with infrared radiation, IR spectra provide a considerable
amount of structural data.
There are four types of instruments for infrared absorption measurements

available:
 Dispersive grating spectrophotometers for qualitative measurements.

 Non-dispersive photometers for quantitative determination of organic
species in the atmosphere
 Fourier transform infrared (FT-IR) instruments for both qualitative and
quantitative measurements.
Infrared spectrometry - Infrared light sources
Instruments for measuring infrared absorption all require a source of

continuous infrared radiation and a sensitive infrared transducer, or detector.
Infrared sources consist of an inert solid that is electrically heated to a

temperature between 1,500 and 2,200 °K. The heated material will then emit
infra-red radiation.
The Nernst glower
The Nernst glower is constructed of rare earth oxides in the form of a hollow
cylinder. Platinum leads at the ends of the cylinder permit the passage of
electricity. Nernst glowers are fragile. They have a large negative temperature
coefficient of electrical resistance and must be preheated to be conductive.

The globar source
A globar is a rod of silicon carbide (5 mm diameter, 50 mm long) which is

electrically heated to about 1,500 °K. Water cooling of the electrical contacts
is needed to prevent arcing. The spectral output is comparable with the Nernst
glower, except at short wavelengths (less than 5 mm) where its output
becomes larger.
The carbon dioxide laser
A tunable carbon dioxide laser is used as an infrared source for monitoring

certain atmospheric pollutants and for determining absorbing species in
aqueous solutions.
Infrared Spectrometry - Detectors
The detectors can be classified into three categories, thermal detectors,

pyroelectric detectors and photo conducting detectors.
Thermal detectors
Thermal detectors can be used over a wide range of wavelengths and they
operate at room temperature. Their main disadvantages are slow response
time and lower sensitivity relative to other types of detectors.
Thermocouple
A thermocouple consists of a pair of junctions of different metals; for example,

two pieces of bismuth fused to either end of a piece of antimony. The potential
difference (voltage) between the junctions changes according to the difference
in temperature between the junctions. Several thermocouples connected in
series are called a thermopile.
Bolometer
A bolometer functions by changing resistance when heated. It is constructed

of strips of metals such as platinum or nickel or from a semiconductor.

Pyroelectric detectors
Pyroelectric detectors consists of a pyroelectric material which is an insulator

with special thermal and electric properties. Triglycine sulphate is the most
common material for pyroelectric infrared detectors. Unlike other thermal
detectors the pyroelectric effect depends on the rate of change of the detector
temperature rather than on the temperature itself. This allows the pyroelectric
detector to operate with a much faster response time and makes these
detectors the choice for Fourier transform spectrometers where rapid response
is essential.
Photo conducting detectors
Photo conducting detectors are the most sensitive detectors. They rely on
interactions between photons and a semiconductor. The detector consists of
a thin film of a semiconductor material such as lead sulphide, mercury
cadmium telluride or indium antimonide deposited on a nonconducting glass
surface and sealed into an evacuated envelope to protect the semiconductor
from the atmosphere. The lead sulphide detector is used for the near-infrared
region of the spectrum. For mid- and far-infrared radiation the mercury
cadmium telluride detector is used. It must be cooled with liquid nitrogen to
minimize disturbances.
SAMPLE HANDLING TECHNIQUES
Sample handling is considered as an important technique in infrared

spectroscopy. There are various methods of sample preparation to enable
almost any type of sample to be examined. Some significant problems arise
when trying to construct sample containers for vibrational spectrometry,
because every material has some vibrational absorption. The material that has
a minimum interference in the regions of interest is used as sample. The
material of choice for IR spectroscopy is a solid potassium bromide. Such
plates are used in a number of ways. Polyethylene pellets are used for
recording the far IR spectra.

Solids
Solids are sampled in a wide variety of ways. If the sample is soluble, it may
be dissolved and handled as for a liquid. Solid samples for which no solvent is
suitable can be prepared for analysis by incorporating them into a pressed
pellet of alkali halide, usually potassium bromide. The sample is mixed with a
weighed amount of powdered potassium bromide and the mixture is admitted
to a pressure of several tones in a die, to produce a highly transparent pellete
or disc which can be inserted into the spectrophotometer. The use of KBr
eliminates the problems of additional bands due to mulling agent. KBr does
not absorb infrared light in the region 2.5–15 m and a complete spectrum of
the sample is obtained. Solid samples have also been examined in the form of
a thin layer deposited by sublimation or solvent evaporation on the surface of
a salt plate. Another method, called mulling has also been developed, in which
the powdered sample is mixed to form a paste with little heavy paraffin oil.
The mull is sandwiched between salt plates for measurement. Mulls are
formed by grinding 2 to 5 mg of finely powered sample in the presence of one
or two drops of a heavy hydrocarbon oil called Nujol.
Liquids
The spectra of a pure liquid can be measured as very thin films squeezed
between two alkali halide windows of a demountable cell. This technique can
produce a film of thickness 0.01 mm or less. The cells are then taken apart
and cleaned. This method is most useful for qualitative work only because the
sample thickness cannot be controlled. For quantitative work sealed liquid
cells of fixed path length in the range 0.01 to 0.1mm are used. Liquid cells
consist of two alkali halide windows usually NaCl or KBr, separated by a
spacer of suitable thickness made of Teflon or lead which limits the volume of
the cell.
Gases
The vapor is introduced into a special cell, usually about 10cm long that can
be placed directly in the path of one of the infrared beams. The end walls of

the cell are usually made of sodium chloride, which is transparent to infrared.
Most organic compounds have too low a vapor pressure for this phase to be
useful. The low frequency vibrational changes in the gaseous phase often split
the high frequency vibrational bands.
Solvents
Solvents of good infrared transparency over a convenient frequency range are

available and the spectra of the sample dissolved in carbon tetrachloride and
carbon disulphide provide the complete range. Chloroform is considered to be
an important solvent and is frequently used because it shows absorptions
though it has less symmetric molecule than carbon tetrachloride and carbon
disulphide.
FTIR-Fourier Transform Infrared Spectrometer
FT-IR stands for Fourier Transform Infrared, the preferred method of infrared
spectroscopy. In infrared spectroscopy, IR radiation is passed through a
sample. Some of the infrared radiation is absorbed by the sample and some of
it is passed through (transmitted). The resulting spectrum represents the
molecular absorption and transmission, creating a molecular fingerprint of
the sample. Like a fingerprint no two unique molecular structures produce
the same infrared spectrum. This makes infrared spectroscopy useful for
several types of analysis.
Fourier Transform Infrared (FT-IR) spectrometry was developed in order to

overcome the limitations encountered with dispersive instruments. The main
difficulty was the slow scanning process. A method for measuring all of the
infrared frequencies simultaneously, rather than individually, was needed. A
solution was developed which employed a very simple optical device called an
interferometer. The interferometer produces a unique type of signal which has
all of the infrared frequencies “encoded” into it. The signal can be measured
very quickly, usually on the order of one second or so. Thus, the time element
per sample is reduced to a matter of a few seconds rather than several
minutes.

Most interferometers employ a beam splitter which takes the incoming

infrared beam and divides it into two optical beams. One beam reflects off of
a flat mirror which is fixed in place. The other beam reflects off of a flat mirror
which is on a mechanism which allows this mirror to move a very short
distance (typically a few millimeters) away from the beam splitter.
The two beams reflect off of their respective mirrors and are recombined
when they meet back at the beam splitter. Because the path that one beam
travels is a fixed length and the other is constantly changing as its mirror
moves, the signal which exits the interferometer is the result of these two
beams “interfering” with each other. The resulting signal is called an
interferogram which has the unique property that every data point (a function
of the moving mirror position) which makes up the signal has information
about every infrared frequency which comes from the source. This means that
as the interferogram is measured, all frequencies are being measured
simultaneously.

Thus, the use of the interferometer results in extremely fast measurements.

Because the analyst requires a frequency spectrum (a plot of the intensity at
each individual frequency) in order to make an identification, the measured
interferogram signal cannot be interpreted directly. A means of “decoding” the
individual frequencies is required. This can be accomplished via a well-known
mathematical technique called the Fourier transformation. This
transformation is performed by the computer which then presents the user
with the desired spectral information for analysis.
Advantages of FTIR
The main advantages of FT spectroscopy are the greater ease and speed of
measurement. The entire spectrum can be recorded within few seconds using
sophisticated computers. Recent developments in FT infrared spectrometers
have thus led to higher resolution, total wavelength coverage, higher accuracy
in frequency and intensity measurements. It can also be used in the
characterization of all kinds of samples. In FT method, all the source energy
passes through the instrument and the resolving power is constant over the
entire spectrum. The signal to noise ratio is also improved. The smoothening
of peaks and the vertical and horizontal expansion of selective region is also
possible.
There are three principal advantages for an FT spectrometer compared to a

scanning (dispersive) spectrometer.
1. The multiplex /Fellgett's advantage
This arises from the fact that information from all wavelengths is collected
simultaneously. It results in a higher signal-to-noise ratio for a given scan-
time for observations limited by a fixed detector noise contribution (typically
in the thermal infrared spectral region where a photodetector is limited by
generation-recombination noise).
2. The throughput /Jacquinot's advantage

This results from the fact that in a dispersive instrument, the monochromator
has entrance and exit slits which restrict the amount of light that passes

through it. The interferometer throughput is determined only by the diameter

of the collimated beam coming from the source. Although no slits are needed,
FTIR spectrometers do require an aperture to restrict the convergence of the
collimated beam in the interferometer. This is because convergent rays are
modulated at different frequencies as the path difference is varied. Such an
aperture is called a Jacquinot stop. For a given resolution and wavelength this
circular aperture allows more light through than a slit, resulting in a higher
signal-to-noise ratio.
3. The wavelength accuracy or Connes' advantage
The wavelength scale is calibrated by a laser beam of known wavelength that

passes through the interferometer. This is much more stable and accurate
than in dispersive instruments where the scale depends on the mechanical
movement of diffraction gratings. In practice, the accuracy is limited by the
divergence of the beam in the interferometer which depends on the resolution.
Another minor advantage is less sensitivity to stray light that is radiation of

one wavelength appearing at another wavelength in the spectrum. In
dispersive instruments, this is the result of imperfections in the diffraction
gratings and accidental reflections. In FT instruments there is no direct
equivalent as the apparent wavelength is determined by the modulation
frequency in the interferometer.
Applications of Infrared spectroscopy
Identification of functional group and structure elucidation
Entire IR region is divided into group frequency region and fingerprint region.
Range of group frequency is 4000-1500 cm-1 while that of finger print region
is 1500-400 cm-1. In group frequency region, the peaks corresponding to
different functional groups can be observed. According to corresponding
peaks, functional group can be determined. Each atom of the molecule is
connected by bond and each bond requires different IR region so characteristic
peaks are observed. This region of IR spectrum is called as finger print region
of the molecule. It can be determined by characteristic peaks.

Identification of substances
IR spectroscopy is used to establish whether a given sample of an organic

substance is identical with another or not. This is because large number of
absorption bands is observed in the IR spectra of organic molecules and the
probability that any two compounds will produce identical spectra is almost
zero. So if two compounds have identical IR spectra then both of them must
be samples of the same substances. IR spectra of two enantiomeric compound
are identical. So IR spectroscopy fails to distinguish between enantiomers.
Studying the progress of the reaction
Progress of chemical reaction can be determined by examining the small

portion of the reaction mixture withdrawn from time to time. The rate of
disappearance of a characteristic absorption band of the reactant group
and/or the rate of appearance of the characteristic absorption band of the
product group due to formation of product is observed.
Detection of impurities
IR spectrum of the test sample to be determined is compared with the

standard compound. If any additional peaks are observed in the IR spectrum,
then it is due to impurities present in the compound.
Quantitative analysis
The quantity of the substance can be determined either in pure form or as a

mixture of two or more compounds. In this, characteristic peak corresponding
to the drug substance is chosen and log I0/It of peaks for standard and test
sample is compared. This is called base line technique to determine the
quantity of the substance.

 Explain in brief principle of Infrared spectroscopy.

 What is dipole moment? Give its importance.
 Give classification of fundamental vibrations.
 What are overtone and combination bands?
 What are coupled vibrations? Give example.
 What are advantages of FTIR?
 What is Hook’s law? Give its importance.

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EXERCISE NO 5 Date:

AIM: To study Jasco FTIR 4200
Reference: Jasco FTIR 4200 Manual
Instrument: Fourier Transform Infrared Spectrometer
Make: Jasco
Model: 4200
Software: Spectra Manager
Specifications:
Parameter Specifications
Standard Wavenumber
7,800 to 350 cm-1
Measurement Range
Wavenumber Accuracy Within ± 0.01 cm-1 (theoretical value)
Maximum Resolution 0.5 cm-1
Optical System Single beam
45º Michelson interferometer

Corner cube mirror interferometer, with auto-
Interferometer
alignment
mechanism, sealed structure
Mirror Coating Aluminum
Standard: Ge/KBr
Beam Splitter
Option: Si/CaF2, Ge/CsI (not interchangeable)
Standard: High-intensity ceramic source

Light Source
Option: Halogen lamp
Deuterated Lanthanum α Alanine doped TriGlycine

Detector
Sulphate ( DLaTGS ) Pyroelectric detector
Signal-to-Noise Ratio
30,000:1
Software Spectra Manager
Operating Procedure

 Power on the spectrometer. When you hear 3 beeps the instrument is

ready.
 Double click the ‘spectra manager’ icon to start the software.
 When the application software is started you will see a menu on the left
side. Choose Spectra measurement.
 Choose B monitor for background correction. Ensure that the sample
chamber is clean and empty.
 Place the sample KBr pellet carefully in the sample chamber.
 Click on Sample monitor.
 Save the spectra.
 Remove the sample pellet.
 Thoroughly clean the sample chamber.
 Power off the spectrometer.
 Shut down the computer.
KBr Pellet Preparation
1. Heat the solid powder sample in oven at about 100°C for 10 min. to
remove moisture.
2. Keep the sample in desiccator.
3. Carefully take sample and KBr powder (1:99) and mix well in mortar.
4. Place the powder in hydraulic press and apply pressure to make a
thin pellet.
5. Remove the pellet with forceps and place in sample holder.
Result: The features of Jasco FTIR were studied.

AIM: To study the interpretation of IR spectra
Reference
1. Silverstein, R.M. and Bassler, G.C., 1963. Spectrometric identification of

organic compounds.
2. Pavia D.L., Lampman G.M., Kriz G.S. and Vyvyan J.A., 2008. Introduction
to spectroscopy, Cengage Learning.
Interpretation of IR spectra
A look at the IR spectrum or values of all possible peaks presents us a vast

number of peaks to interpret. However, IR spectrum has to be searched for
the presence or absence of functional groups in particular regions. While
interpreting the IR spectrum, the following facts should be kept in mind.
 An IR spectrum only gives functional group information and it does not

provide a complete structural formula.
 There are only a few regions in an IR spectrum, which provide useful
information. Therefore, assigning all the bands in a spectrum is not
informative and it is not necessary to interpret every peak.
 The presence or absence of a band in each important region is indicative of
a certain functional group.
 To interpret an IR spectrum, examine and divide in the IR spectrum into
different zones, based upon the energy absorbed by common functional
groups. These zones are as follows –

ZONE 1 (3700-3200 cm-1)
Alcohol O-H, Amide/amine N-H, Alkyne ≡ CH
 Alcohol OH : Usually the broadest peak in the spectrum, 3650-3200 cm-1

 Broadening of peak increases if –OH and –NH both are present. This is
because of hydrogen bonding between them.
 Amine /amide NH : usually a broad peak , 3500-3300 cm-1
 Alkyne ≡ CH : Strong peak at ~ 3300 cm-1
ZONE 2 (3200-3700 cm-1)
Aryl/Alkyl CH, Aldehyde CH, Carboxylic acid OH
 Alkyl CH: Peaks right to 3000 cm-1

 Aryl/Vinyl CH : Peaks left to 3000 cm-1
 Aldehyde CH : Two peaks at 2700 and 2900 cm-1
 Carboxylic acid OH : Broad peak 3000-2500 cm-1
ZONE 3 (2300-2000 cm-1)
Alkyne and Nitrile triple bonds

 It is difficult to distinguish these two types of peaks from just the

stretching frequency. The absence or presence of Nitrogen can be
confirmed by NMR data.
ZONE 4 (1850-1650 cm-1)
Carbonyl functional group
 Carbonyl C=O produces the most intense peak of any functional groups
in five zones.
 Aromatic overtone peaks usually appear in this zone between 1950-1750
cm-1 as 4 small bumps.
 They indicate the presence of benzene ring.
ZONE 5 (1650-1400 cm-1)
Alkene C=C, Benzene C=C
 Benzene C=C: Shows two peaks at 1600 and 1500 cm-1.

 Alkene C=C: Peak between 1680 and 1620 cm-1.
ZONE 6 (1300-1400 cm-1)
Fingerprint region
STEPS FOR INTERPRETATION OF IR SPECTRA
STEP I: Look first for carbonyl C=O bond
Carbonyl C=O usually gives the most intense and sharp peak in the spectrum.
Following are the different functional groups containing carbonyl C=O and
corresponding frequencies at which it usually show absorbance.

FREQUENCY AT WHICH C=O

FUNCTIONAL GROUP
SHOWS ABSORBANCE
Acid Halide R-CO-Cl 1800 cm-1
Anhydride 1810 and 1760 cm-1
Ester 1735 cm-1
Aldehyde 1725 cm-1
Ketone 1715 cm-1
Carboxylic acid 1710 cm-1
Amide 1690 cm-1
Confirmation:
Aldehyde
1. Look for C-H peaks in Zone II (2700 – 3200 cm-1 )

2. Aldehydes give two peaks at 2700 cm-1 and 2900 cm-1
3. C=O peak at ~ 1725 cm-1
Ketone
1. Carbonyl C=O peak near 1700 -1715 cm-1
2. Absence of aldehyde –CH two peaks
Acid
1. OH broad peak in Zone II (3300-2500 cm-1)

2. C=O peak near 1710 cm-1

3. C-O peak in 1300-1000 cm-1
Ester
1. C=O peak at ~ 1735 cm-1

2. C-O peak in 1300-1000 cm-1
3. Absence of OH broad peak
Anhydride
1. C=O peaks at 1810 and 1760 cm-1

Amide
1. Sharp peak in zone I (3300-3500 cm-1 )

2. C=O at 1690 cm-1
STEP II: Look for OH bond
If no Carbonyl bond frequency seen in the spectrum, then look for and
alcohol –OH bond and C-O single bond. It can be alcohol/ether
Alcohol
1. Broad OH peak in Zone I (3650-3200 cm-1)

2. C-O peak between 1300-1000 cm-1
Ether
1. C-O stretch near 1300-1000 cm-1

STEP III: Look for double bond C=C (Aromatic/alkene)
Aromatic
1. Look for benzene C=C, two peaks at 1600 and 1500 cm-1
2. CH stretching peaks in Zone II ( Left to 3000 cm-1)
3. Aromatic overtones in Zone IV (1950 TO 1750 cm-1) as 4 small bumps

4. Aromatic ring CH out of plane bending peaks in 900 – 675 cm-1
Alkene
1. Weak absorption by C=C between 1680 – 1620 cm-1

2. CH stretching peaks in Zone II ( Right to 3000 cm-1)
STEP IV: Look for Triple bond
Alkyne
1. A sharp peak near 2150 cm-1

2. Alkyne CH stretch in Zone I, ~ 3300 cm-1
Cyanide
1. Sharp peak near 2250 cm-1

2. NH peak in zone I
STEP V: If no functional group detected then look for Alkane CH peaks

(Right to 3000 cm-1)
STEP VI: If still the structure cannot be assigned, look for halide peaks in
fingerprint region.
Halide Bond Frequency observed
C-Cl 850 – 550 cm-1
C-Br 690 – 515 cm-1
C-I 600 - 500 cm-1
C-F 1400 - 1000 cm-1
Result: The steps for interpretation of IR spectra were studied.

 What are the different zones for IR spectra interpretation?

 Give applications of IR spectroscopy.
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AIM: To study the interpretation of IR spectra of aldehyde

group containing compounds.
Reference

organic compounds.
2. Pavia, D.L., Lampman, G.M., Kriz, G.S. and Vyvyan, J.A., 2008.
Introduction to spectroscopy. Cengage Learning.
3. http://www.orgchemboulder.com/Spectroscopy/irtutor
Requirements:
Chemicals: KBr, Benzaldehyde
Apparatus: FTIR accessories
Instrument: Jasco FTIR
Reference IR spectra of Benzaldehyde

Interpretation
Reference IR spectra of Butyraldehyde

Interpretation
Recorded spectra

Result: The IR spectra interpretation of Aldehyde group containing

compounds was studied.
 How do you confirm presence of aldehyde group in compound from IR

spectra?
 How do you differentiate between aldehyde and ketone by IR spectral
interpretation?
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AIM: To study the interpretation of IR spectra of alcohol group

containing compounds.
Reference

organic compounds.
Requirements:
Chemicals: KBr, Ethanol
Reference IR spectra of Ethanol

Interpretation
Recorded spectra

Reference spectra of Isopropanol
Interpretation

Recorded spectra

Result: The IR spectra interpretation of Alcohol group containing compounds

was studied.
 How do you confirm presence of alcohol group in compound from IR

spectra?
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AIM: To study the interpretation of IR spectra of carboxylic

acid group containing compounds.
Reference:

organic compounds.

Requirements:
Chemicals: KBr, Hexanoic acid
Reference IR spectra of Hexanoic Acid
Interpretation

Recorded spectra

Result: The IR spectra interpretation of Carboxylic acid group containing

compounds was studied.
 How do you confirm presence of carboxylic acid in compound from IR

spectra?
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AIM: To study the interpretation of IR spectra of alkyne group

Reference:

organic compounds.
Requirements:
Chemicals: KBr, 1 Hexyne
Reference IR spectra of 1Hexyne

Interpretation

Result: The IR spectra interpretation of Alkyne group containing compounds

was studied.
 How do you confirm presence of alkyne group in compound from IR

spectra?

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AIM: To study the interpretation of IR spectra of Ester group

Reference:

organic compounds.
Requirements
Chemicals: KBr, Ethyl benzoate, Ethyl Acetate
Reference IR spectra of Ethyl Benzoate

Interpretation
Reference IR spectra of Ethyl Acetate

Interpretation
Recorded spectra

Result: The IR spectra interpretation of Ester group containing compounds

was studied.
 How do you confirm presence of ester group in compound from IR

spectra?

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AIM: To study the interpretation of IR spectra of Ketone group

Reference:

organic compounds.
Requirements:
Chemicals: KBr, 2 Butanone
Reference IR spectra of 2-Butanone

Interpretation

Recorded spectra
Result: The IR spectra interpretation of Ketone group containing compounds

was studied.
 How do you confirm presence of Ketone group in compound from IR

spectra?

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AIM: To study the interpretation of IR spectra of Amine group

Reference:

organic compounds.
Requirements:
Chemicals: KBr, Aniline
Reference IR spectra of Aniline – Primary Amine

Interpretation
This primary amine shows two N–H stretches (3442, 3360); note the shoulder
band, which is an overtone of the N–H bending vibration. The C–N stretch
appears at 1281 rather than at lower wavenumbers because aniline is an
aromatic compound. Also note the N–H bend at 1619.
Reference IR spectra of Diethyl amine –Secondary Amine
Interpretation
Note that this secondary amine shows only one N–H stretch (3288). The C–N
stretch is at 1143, in the range for non-aromatic amines (1250-1020). Diethyl
amine also shows an N–H wag (733).

Reference spectra of Triethylamine –Tertiary amine
Interpretation
Triethylamine is a tertiary amine and does not have an N–H stretch, nor an N–
H wag. The C–N stretch is at 1214 cm-1 (non-aromatic).

Recorded spectra
Result: The IR spectra interpretation of Amine group containing compounds

was studied.
 How do you confirm presence of Amine group in compound from IR

spectra?
 How can we differentiate between primary, secondary and tertiary amine
for IR spectra?

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AIM: To study the interpretation of IR spectra of Aromatic

compound.
Reference:

organic compounds.
Requirements:
Chemicals: KBr, Toulene

Reference IR spectra of Toulene
Interpretation

Recorded spectra
Result: The IR spectra interpretation of Aromatic compounds was studied.
 How do you confirm presence of aromatic ring in compound from IR

spectra?

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AIM: To perform Q-Analysis method for estimation of

Paracetamol and Aceclofenac in bulk mixture.
Reference:

2. Pawar, V.T., Pishawikar, S.A. and More, H.N., 2010. Spectrophotometric
estimation of aceclofenac and paracetamol from tablet dosage form.
Current Pharma Research, 1(1), p.25.
571.
Requirements:
1. Apparatus-: Glass beakers, volumetric flask, funnel, graduated pipette,

etc.
2. Chemicals -: Paracetamol, Aceclofenac, Methanol (AR Grade) etc.
3. Instrument-: UV Visible Spectrophotometer, Sonicator.
Theory
Refer exercise no 3 for theory of Q-Analysis method.

Procedure


Pipette out 1 ml from Paracetamol stock solution and 1 ml from Aceclofenac

5 bulk mixtures.
Observations:

of absorptivities


Average Average
ax1 = ax2 =

of absorptivities

Average Average
ay1 = ay2 =
Absorbance of bulk mixture

No. A1 A2
Calculations


Result
DRUG
Deviation Found
(µg/ml) (µg/ml)
Paracetamol (X)
Aceclofenac (Y)

 What is Q-point?
 How do you select wavelengths in Q-analysis method?

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AIM: To perform Q -Analysis method for estimation of

Paracetamol and Aceclofenac in tablet mixture.
Reference

2. Pawar, V.T., Pishawikar, S.A. and More, H.N., 2010. Spectrophotometric
estimation of aceclofenac and paracetamol from tablet dosage form.
Current Pharma Research, 1(1), p.25.
571.
Requirements

etc.
Theory

7) Uses- Rheumatiod arthritis osteoarthritis.
Procedure


tablet containing 10 mg of Aceclofenac and 32.5 mg of Paracetamol. Dissolve
the equivalent weight in 100 ml of Methanol. Sonicate for 10 min and filter
through the Whatmann filter. This gives a tablet stock solution containing 325
µg/ml of Aceclofenac and 100 µg/ml of Paracetamol. Pipette out 0.5 ml of
tablet stock solution and dilute up to 10 ml with distilled water. Prepare such
5 samples.
Observations

of absorptivities


Average Average
ax1 = ax2 =

of absorptivities

Average Average
ay1 = ay2 =

No. A1 A2

Calculations

Result
Standard %
Deviation Assay
(µg/ml) (µg/ml)
Paracetamol (X)
Aceclofenac (Y)


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AIM: To perform Q Analysis method for estimation of

Paracetamol and Ibuprofen in Bulk mixture.
Reference

2. Joshi, R.S., Pawar, N.S., Katiyar, S.S., Zope, D.B. and Shinde, A.T., 2011.
Development and validation of UV spectrophotometric methods for
simultaneous estimation of Paracetamol and Ibuprofen in pure and tablet
dosage form. Der pharmacia sinica, 2(3), pp.164-171.
571.
Requirements

etc.
Theory

Ibuprofen – Drug Profile
1) Name- Ibuprofen


injury.

alpha-methylacyl-CoA racemase. In particular, it is generally proposed that
the R-enantiomer
Procedure

Pipette out 1 ml from Paracetamol stock solution and 1 ml from Ibuprofen

5 bulk mixtures.

Observations

of absorptivities

Average Average
ax1 = ax2 =

absorptivities

Average Average
ay1 = ay2 =


No. A1 A2
Calculations

Result
DRUG
Deviation Found
(µg/ml) (µg/ml)
Paracetamol (X)
Ibuprofen (Y)

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AIM: To perform Q Analysis method for estimation of

Paracetamol and Ibuprofen in Tablet mixture.
Reference

2. Joshi, R.S., Pawar, N.S., Katiyar, S.S., Zope, D.B. and Shinde, A.T., 2011.
Development and validation of UV spectrophotometric methods for
simultaneous estimation of Paracetamol and Ibuprofen in pure and tablet
dosage form. Der pharmacia sinica, 2(3), pp.164-171.
571.
Requirements
1. Apparatus-: Glass beakers, volumetric flask, funnel, graduated pipette,

etc.
2. Chemicals -: Paracetamol, Ibuprofen, Methanol (AR Grade) etc.
3. Instrument-: UV Visible Spectrophotometer, Sonicator.
Theory

Ibuprofen – Drug profile
1) Name- Ibuprofen


injury.

alpha-methyl acyl-CoA racemase. In particular, it is generally proposed that
the R-enantiomer
Procedure

tablet containing 10 mg of Ibuprofen and 8.125 mg of Paracetamol. Dissolve
in 100 ml of Methanol. Sonicate for 10 min and filter through the Whatmann

filter. This gives a tablet stock solution containing 100 µg/ml of Ibuprofen and
8 µg/ml of Paracetamol.
Pipette out 1 ml of tablet stock solution and dilute up to 10 ml with distilled

water. Prepare such 5 samples.
Observations

of absorptivities

Average Average
ax1 = ax2 =

absorptivities


Average Average
ay1 = ay2 =

No. A1 A2
Calculations

Result
Standard %
Deviation Assay
(µg/ml) (µg/ml)
Paracetamol (X)
Ibuprofen (Y)


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EXERCISE NO 6 Date:
Aim: To study principle, instrumentation and applications of

Gas chromatography.
References:
1. Willard, H.H., Merritt Jr, L.L., Dean, J.A. and Settle Jr, F.A., 1988.
Instrumental methods of analysis.
2. https://nptel.ac.in/courses/103108100/module7/module7.pdf
3. https://en.wikipedia.org/wiki/Gas_chromatography#Carrier_gas_selec
tion_and_flow_rates
Introduction
Gas chromatography differs from other forms of chromatography in that the

mobile phase is a gas and the components are separated as vapours. It is thus
used to separate and detect small molecular weight compounds in the gas
phase. The sample is either a gas or a liquid that is vaporized in the injection
port. The mobile phase for gas chromatography is a carrier gas, typically
helium because of its low molecular weight and being chemically inert.
Pressure is applied and the mobile phase moves the analyte through the
column. The separation is accomplished using a column coated with a
stationary phase.
Principle
The equilibrium for gas chromatography is partitioning, and the components

of the sample will partition (i.e. distribute) between the two phases: the
stationary phase and the mobile phase. Compounds that have greater affinity
for the stationary phase spend more time in the column and thus elute later
and have a longer retention time (Rt) than samples that have higher affinity
for the mobile phase.

Affinity for the stationary phase is driven mainly by intermolecular

interactions and the polarity of the stationary phase can be chosen to
maximize interactions and thus the separation. Ideal peaks are Gaussian
distributions and symmetrical, because of the random nature of the analyte
interactions with the column. The separation is hence accomplished by
partitioning the sample between the gas and a thin layer of a non-volatile
liquid held on a solid support.
A sample containing the solutes is injected onto a heated block where it is

immediately vaporized and swept as a plug of vapour by the carrier gas stream
into the column inlet. The solutes are adsorbed by the stationary phase and
then desorbed by fresh carrier gas. The process is repeated in each plate as
the sample is moved toward the outlet. Each solute will travel at its own rate
through the column. Their bands will separate into distinct zones depending
on the partition coefficients, and band spreading. The solutes are eluted one
after another in the increasing order of their Kd, and enter into a detector
attached to the exit end of the column.
Here they register a series of signals resulting from concentration changes and
rates of elution on the recorder as a plot of time versus the composition of
carrier gas stream. The appearance time, height, width and area of these peaks
can be measured to yield quantitative data.

Instrumentation
Gas chromatography is mainly composed of the following parts:
1. Carrier gas in a high pressure cylinder with attendant pressure

regulators and flow meters
 Helium, N2, H, Argon are used as carrier gases.
 Helium is preferred for thermal conductivity detectors because of its

high thermal conductivity relative to that of most organic vapours.
 N2 is preferable when large consumption of carrier gas is employed.
 Carrier gas from the tank passes through a toggle valve, a flow meter,
(1-1000 ml / min), capillary restrictors, and a pressure gauge (1-4 atm).
 Flow rate is adjusted by means of a needle valve mounted on the base

of the flow meter and controlled by capillary restrictors.
 The operating efficiency of the gas chromatograph is directly dependant

on the maintenance of a constant gas flow.
2. Sample injection system
 Liquid samples are injected by a micro syringe with needle inserted

through a self-scaling, silicon-rubber septum into a heated metal block
by a resistance heater.
 Gaseous samples are injected by a gas tight syringe or through a by-

pass loop and valves.
 Typical sample volumes range from 0.1 to 0.2 ml.
3. The separation column
 The heart of the gas chromatography is the column which is made of

metals bent in U shape or coiled into an open spiral or a flat pancake
shape.
 Copper is useful up to 2500

 Several sizes of columns are used depending upon the requirements.
4. Liquid phases
 An infinite variety of liquid phases are available limited only by their

volatility, thermal stability and ability to wet the support.
 No single phase will serve for all separation problems at all

temperatures.
Non Polar – Paraffin, squalene, silicone greases, silicone gum rubber. These
materials separate the components in order of their boiling points.
Intermediate Polarity – These materials contain a polar or polarizable group

on a long non polar skeleton which can dissolve both polar and non-polar
solutes. For example- Diethyl hexyl phthalate is used for the separation of
high boiling alcohols.
Polar – Carbowaxes – Liquid phases with large proportion of polar groups.

Separation of polar and non-polar substances.
Hydrogen bonding – Polar liquid phases with high hydrogen bonding e.g.
Glycol.
Specific purpose phases – Relying on a chemical reaction with solute to

achieve separations. For example - AgNO3 in glycol separates unsaturated
hydrocarbons.
5. Supports
The structure and surface characteristics of the support materials are

important parameters, which determine the efficiency of the support and the
degree of separation respectively. The support should be inert but capable of
immobilizing large volume of liquid phase as a thin film over its surface.
Surface area should be large to ensure rapid attainment of equilibrium

between stationary and mobile phases.

Support should be strong enough to resist breakdown in handling and be

capable of packed into a uniform bed. Diatomaceous earth, kiesulguhr treated
with Na2CO3 for 9000 C causes the particle fusion into coarser aggregates.
Glass beads with a low surface area and low porosity can be used to coat up
to 3% stationary phases. Porous polymer beads differing in the degree of cross
linking of styrene with alkyl-vinyl benzene are also used which are stable up
to 2500.
6. Detector
Detectors sense the arrival of the separated components and provide a signal.
These are either concentration dependant or mass dependant. The detector
should be close to the column exit and correct temperature to prevent
decomposition.
As solutes elute from the column, they interact with the detector. The detector
converts this interaction into an electronic signal that is sent to the data
system. The magnitude of the signal is plotted versus time (from the time of
injection) and a chromatogram is generated.
Some detectors respond to any solute eluting from the column while others
respond only to solutes with specific structures, functional groups or atoms
Detectors that exhibit enhanced response to specific types of solutes are called
selective detectors.
Most detectors require one or more gases to function properly. There are
combustion, reagent, auxiliary and makeup gases. In some cases, one gas may
serve multiple purposes. The type of detector gas is dependent on the specific
detector and is fairly universal between GC manufacturers. The flow rates for
each type of detector varies between GC manufacturers. It is important to
follow the recommended flow rates to obtain the optimal sensitivity, selectivity
and linear range for a detector.
Flame ionization detector (FID)

Mechanism: Compounds are burned in a hydrogen-air flame. Carbon

containing compounds produce ions that are attracted to the collector. The
number of ions hitting the collector is measured and a signal is generated.
Selectivity: Compounds with C-H bonds. A poor response for some non-
hydrogen containing organics (e.g., hexachlorobenzene).
Sensitivity: 0.1-10 ng
Linear range: 105-107
Gases: Combustion - hydrogen and air; Makeup - helium or nitrogen

Temperature: 250-300°C, and 400-450°C for high temperature analyses.
Nitrogen phosphorus detector (NPD)
Mechanism: Compounds are burned in a plasma surrounding a rubidium

bead supplied with hydrogen and air. Nitrogen and phosphorous containing
compounds produce ions that are attracted to the collector. The number of
ions hitting the collector is measured and a signal is generated.
Selectivity: Nitrogen and phosphorous containing compounds
Sensitivity:1-10page
Linear range: 104-10-6
Gases: Combustion - hydrogen and air; Makeup - helium
Temperature: 250-300°C
Electron capture detector (ECD)
Mechanism: Electrons are supplied from a 63Ni foil lining the detector cell. A
current is generated in the cell. Electronegative compounds capture electrons
resulting in a reduction in the current. The amount of current loss is indirectly
measured and a signal is generated.
Selectivity: Halogens, nitrates and conjugated carbonyls
Sensitivity: 0.1-10 page (halogenated compounds); 1-100 page
(nitrates); 0.1-1 ng (carbonyls)

Gases: Nitrogen or argon/methane
Thermal conductivity detector (TCD)
Mechanism: A detector cell contains a heated filament with an applied current.

As carrier gas containing solutes passes through the cell, a change in the
filament current occurs. The current change is compared against the current
in a reference cell. The difference is measured and a signal is generated.
Selectivity: All compounds except for the carrier gas
Sensitivity: 5-20 ng
Gases: Makeup - same as the carrier gas
Temperature:150-250°C
Flame photometric detector (FPD)
Mechanism: Compounds are burned in a hydrogen-air flame. Sulphur and

phosphorous containing compounds produce light emitting species (sulphur
at 394 nm and phosphorous at 526 nm). A monochromatic filter allows only
one of the wavelengths to pass. A photomultiplier tube is used to measure the
amount of light and a signal is generated. A different filter is required for each
detection mode.
Selectivity: Sulfur or phosphorous containing compounds. Only one at a time.

Sensitivity: 10-100 page (sulfur); 1-10 page (phosphorous)
Linear range: Non-linear (sulfur); 103-105 (phosphorous)
Gases: Combustion - hydrogen and air; Makeup - nitrogen

Photoionization detector (PID)

Mechanism: Compounds eluting into a cell are bombarded with high energy
photons emitted from a lamp. Compounds with ionization potentials below the
photon energy are ionized. The resulting ions are attracted to an electrode,
measured, and a signal is generated.
Selectivity: Depends on lamp energy. Usually used for aromatics and olefins
(10 eV lamp).
Sensitivity: 25-50 page (aromatics); 50-200 page (olefins)
Gases: Makeup - same as the carrier gas
Temperature: 200°C
MASS SPECTROMETER (MS)
Mechanism: The detector is maintained under vacuum. Compounds are

bombarded with electrons (EI) or gas molecules (CI). Compounds fragment
into characteristic charged ions or fragments. The resulting ions are focused
and accelerated into a mass filter. The mass filter selectively allows all ions of
a specific mass to pass through to the electron multiplier. All of the ions of the
specific mass are detected. The mass filter then allows the next mass to pass
through while excluding all others. The mass filter scans stepwise through the
designated range of masses several times per second. The total number of ions
are counted for each scan. The abundance or number of ions per scan is
plotted versus time to obtain the chromatogram (called the TIC). A mass
spectrum is obtained for each scan which plots the various ion masses versus
their abundance or number.
Selectivity: Any compound that produces fragments within the selected mass
range. May be an inclusive range of masses (full scan) or only select ions (SIM).
Sensitivity: 1-10 ng (full scan); 1-10 page (SIM)
Gases: None

Temperature: 250-300°C (transfer line), 150-250°C (source)
7. Recorder
The recorder should be generally 10 mv (full scale) fitted with a fast response
pen (1 sec or less). The recorder should be connected with a series of good
quality resistances connected across the input to attenuate the large signals.
An integrator may be a good addition.
Procedure of Gas Chromatography
Step 1: Sample Injection and Vaporization
1. A small amount of liquid sample to be analysed is drawn up into a syringe.
2. The syringe needle is positioned in the hot injection port of the gas
chromatograph and the sample is injected quickly.
3. The injection of the sample is considered to be a “point” in time, that is, it

is assumed that the entire sample enters the gas chromatograph at the
same time, so the sample must be injected quickly.
4. The temperature is set to be higher than the boiling points of the

components of the mixture so that the components will vaporise.
5. The vaporised components then mix with the inert gas mobile phase to be
carried to the gas chromatography column to be separated.
Step 2: Separation in the Column
1. Components in the mixture are separated based on their abilities to adsorb

on, or bind to, the stationary phase.
2. A component that adsorbs most strongly to the stationary phase will spend
the most time in the column (will be retained in the column for the longest
time) and will therefore have the longest retention time (Rt). It will emerge
from the gas chromatograph last.

3. A component that adsorbs the least strongly to the stationary phase will
spend the least time in the column (will be retained in the column for the
shortest time) and will therefore have the shortest retention time (Rt). It will
emerge from the gas chromatograph first.
4. If we consider a 2 component mixture in which component A is

more polar than component B then:
 component A will have a longer retention time in a polar column than

component B
 component A will have a shorter retention time in a non-polar column than

component B
Step 3: Detecting and Recording Results
1. The components of the mixture reach the detector at different times due to
differences in the time they are retained in the column.
2. The component that is retained the shortest time in the column is detected
first. The component that is retained the longest time in the column is
detected last.
3. The detector sends a signal to the chart recorder which results in a peak on
the chart paper. The component that is detected first is recorded first. The
component that is detected last is recorded last.
Applications of Gas Chromatography
 GC analysis is used to calculate content of a chemical product, for example

in assuring the quality of products in the chemical industry; or measuring
toxic substances in soil, air or water.
 Gas chromatography is used in the analysis of Air-borne pollutants,

Performance enhancing drugs in athletes urine samples, oil spills and
essential oils in perfume preparation

 GC is very accurate if used properly and can measure Pico moles of a

substance in a 1 ml liquid sample, or parts-per-billion concentrations in
gaseous samples.
 Gas Chromatography is used extensively in forensic science. Disciplines as

diverse as solid drug dose (pre-consumption form) identification and
quantification, arson investigation, paint chip analysis, and toxicology cases,
employ GC to identify and quantify various biological specimens and crime-
scene evidence.
 What is Gas Chromatography?

 Give applications of Gas Chromatography.
 Enlist different detectors used in Gas chromatography.
 Give applications of Gas chromatography.
 Write Van-Deemter equation for gas chromatography.
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FINAL YEAR B. PHARM.

SEM VII PHARMACEUTICAL ANALYSIS V

Aim: To study principle, instrumentation and applications of

Spectroscopic analysis is a study of interaction of light or radiation with

According to photon theory, the light is made up of energy packets. These

Electro-magnetic radiation theory

The properties of electromagnetic radiation are conveniently described by

SCES’S INDIRA COLLEGE OF PHARMACY, PUNE

Amplitude: It is the length of the electric vectors at a maximum in the wave.

Wavelength: It is the linear distance between any 2 equivalent points on

Wave No.: It is the reciprocal of wavelength is cms unit

Constructive Interference: A maximum amplitude occurs when the two

Destruction Interference: The resultant amplitude is smaller than the

λ max : The wavelength which is absorbed maximum by a molecule is called

as wavelength of maximum absorption. (λ max)

INTERACTION OF MOLECULE WITH RADIATION

The energy of the radiation in the visible range is generally: 36 to 72 kcal/mole

SCES’S INDIRA COLLEGE OF PHARMACY, PUNE

The electrons in a molecule can be of one of three types: namely σ (single

3. n-electrons or non-bonding electrons are generally electrons belonging to

Most of the absorption in the ultraviolet-visible spectroscopy occurs due to π-

SCES’S INDIRA COLLEGE OF PHARMACY, PUNE

Selection rules for electronic transitions

π to π* transition is symmetry allowed - High intensity with ε more than 1000

n to π* transition is symmetry forbidden – low intensity with ε less than 100

Transition among electronic states of same spin multiplicity are allowed. If

BEER LAMBERT’S LAW

1) Beers law states that intensity of a beam of parallel monochromatic

2) Lamberts law states that intensity of a beam of parallel monochromatic

A combination of the two Law yields the Beer’s-Lamberts Law

SCES’S INDIRA COLLEGE OF PHARMACY, PUNE

IT = Transmitted light intensity

1gm/100ml in a 1cm cell.

Relationship between Molar absorptivity and Specific absorbance

Concept of Chromophore and Auxochrome in the UV spectroscopy

SCES’S INDIRA COLLEGE OF PHARMACY, PUNE

Solvent UV Cut off wavelength

SCES’S INDIRA COLLEGE OF PHARMACY, PUNE

SCES’S INDIRA COLLEGE OF PHARMACY, PUNE

SCES’S INDIRA COLLEGE OF PHARMACY, PUNE

the far UV region. Most monochromators use gratings though. Some

The slit of a monochromator play an important role in determining the

SCES’S INDIRA COLLEGE OF PHARMACY, PUNE

The entrance slit of a monochromator serves as a radiation source, its image

Movement of monochromator setting in one direction or the other produces a

Selection of slit width

The effective bandwidth of a monochromator depends on the dispersion of the

SCES’S INDIRA COLLEGE OF PHARMACY, PUNE

(2) Quartz or fused silica can be used as material of construction of cuvette

Transducers convert radiant energy into electrical energy. An ideal transducer

S = current /voltage output

Many real transducers exhibit a small constant response known as ‘Dark

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K d = dark current which is usually constant over a short measurement

Types of radiation transducers

(a) Transducers which respond to heat

The thermal transducers respond to average power of the incident radiation.

Different photon transducers in use are as follows-

(1) Photovoltaic cell, in which radiant energy generates current at the

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(6) Charge transfer transducers in which the charges developed in a silicon

It consist of a flat copper or iron electrode on which it deposited a layer of

When radiation of sufficient energy reaches the semiconductor, electrons and