Instru booklet

Phram. Chemistry II
Instrumentation

Atomic
absorption
&
Emission
Spectroscopy

Atomic Absorption spectroscopy
The technique was introduced in 1955 by Walsh in
Australia. The first commercial atomic absorption
spectrometer was introduced in 1959.
Definition: When we study the absorption of
energy by the atoms in the flame, we call this
technique as Atomic Absorption Spectroscopy AAS.
Atomic Absorption Spectroscopy (AAS) is an analytical
technique that measures the concentrations of metals
and metalloids in samples. It makes use of the
absorption of light by these elements, in order to
measure their concentration.
Principle:
Atomic-absorption spectroscopy quantifies or measures the
absorption of energy radiation by ground state atoms in the
gaseous state.
The absorption of the ultraviolet or visible light energy that has the
right wavelength causes the electrons of the sample to be
promoted from a lower energy level to a higher energy level. The
analyte concentration is determined from the amount of
absorption.
The change in energy can be calculated as;
= −
=
=
So, the equation will be;
= 
Where;
 ΔE – Change in energy
 E1 – Excited energy
 E0 – ground state
 h – Planck’s constant
 c – velocity of light
  – wavelength

Concentration measurements are usually determined from a working curve after calibrating the
instrument with standards of known concentration.
The basic equation is; ∝
Instrumentation:
Atomic absorption spectrometer have four principal components;
1. A light source (usually a hollow cathode lamp)
2. Beam chopper
3. Atomizer
4. A Monochromator
5. A detector, and read out device
Hollow cathode lamp:
The light source is usually a hollow cathode lamp of the element that is being measured. It contains a
tungsten anode and a hollow cylindrical cathode made of the element to be determined. These are
sealed in a glass tube filled with an inert gas (neon or argon). Each element has its own unique lamp
which must be used for that analysis.

Working:

Applying a potential difference between the anode and the cathode leads to the ionization of some gas
atoms.



These gaseous ions bombard the cathode and eject metal atoms from the cathode in a process
called sputtering. Some sputtered atoms are in excited states and emit radiation, as they fall
back to the ground state.



The shape of the cathode which is hollow cylindrical concentrates the emitted radiation into a beam
which passes through a quartz window all the way to the vaporized sample.



Since atoms of different elements absorb characteristic wavelengths of light. Analyzing a sample to see if
it contains a particular element means using light from that element.

Example:
A lamp containing lead emits light from excited lead atoms that produce the right mix of wavelengths to
be absorbed by any lead atoms from the sample.
A beam of the electromagnetic radiation emitted from excited lead atoms is passed through the
vaporized sample. Some of the radiation is absorbed by the lead atoms in the sample. The greater the
number of atoms there is in the vapor, the more radiation is absorbed.
Beam chopper:
It is present between the hollow cathode lamp and flame. It rotates and breaks the steady light into
intermittent light. This gives a pulsating current in photo cell.
Atomizer:
Elements to be analyzed needs to be in atomic sate.
Atomization refers to the separation of particles into individual molecules and breaking molecules into
atoms .This is done by exposing the analyte to high temperatures in a flame or graphite furnace.
The role of the atom is to primarily dissolvate a liquid sample and then the solid particles are vaporized
into their free gaseous ground state form. In this form atoms will be available to absorb radiation
emitted from the light source and thus generate a measurable signal proportional to concentration.
There are two types of atomization;

Flame atomization


Graphite furnace atomization

Atomization method or energy source is selected according to the sensitivity and selectivity of the sample.


Flame atomization:

Flame Atomic absorption can only analyze liquids and solution samples, where it uses a burner to
increase the path length, and therefore to increase the total absorbance.
Sample solutions are usually introduced into a nebulizer by being sucked up by a capillary tube .In the
nebulizer the sample is dispersed into tiny droplets, which can be readily broken down in the flame.
The fine mist of droplets is mixed with fuel (acetylene), and oxidant (nitrous oxide) and burned. The
flame temperature is important because it influences the distribution of atoms. It can be manipulated by
oxidant and fuel ratio. The technique is thus named as Flame atomic absorption spectroscopy.
Various types of flames used in Atomic absorption Spectroscopy;
Fuel and Oxidant Temperature o
C
Gas : Air 1700o
C – 1900o
C
Gas : Oxygen O2 2700 o
C – 2800 o
C
Hydrogen H2 : Air 2000 o
C – 2100 o
C
Hydrogen H2 : Oxygen O2 2550 o
C – 2700 o
C
Acetylene : Air 2100 o
C – 2400 o
C
Acetylene : Oxygen O2 3050 o
C – 3150 o
C
Acetylene : N2O 2600 o
C –2 800 o
C
Process taking place in flame:
Following is the process that occurs in the
flame;

Nebulization: conversion of
liquid sample into a fine spray.

Desolvation: Solid atoms are
mixed with the gaseous fuel.

Volatilization: Solid atoms are
converted into to a vapor in a
flame.
There are three types of particles that
exist in the flame;
1. Atoms
2. Ions
3. Molecules


Graphite furnace atomization:

Graphite furnace is used for the atomization of the
sample. The sample is dried then burned to ash and
finally atomized. The technique is thus named as
Graphite atomic absorption spectroscopy. This
technique should be used only when the sample size is
small and/ or when a greater sensitivity is needed.
Graphite atomic absorption can analyze liquid, solid,
semi-solid and solution samples. It should not be used
when ordinary flame AA would do as well, since there
are disadvantages relating to sample size and precision.
Monochromator:
This is a very important part in an Atomic Absorption spectrometer. It is used to separate out all of the
thousands of lines. Without a good monochromator, detection limits are severely compromised.
A monochromator is used to select the specific wavelength of light which is absorbed by the sample, and
to exclude other wavelengths. The selection of the specific light allows the determination of the selected
element in the presence of others.
Detector and read out device:
The light selected by the monochromator is directed onto a
detector that is typically a Photomultiplier tube, whose function is
to convert the light signal into an electrical signal proportional to
the light intensity.
The processing of electrical signal is fulfilled by a signal amplifier.
The signal could be displayed for readout, or further fed into a data
station for printout by the requested format.
Calibration curve:
A calibration curve is used to determine the unknown
concentration of an element in a solution. The
instrument is calibrated using several solutions of
known concentrations. The absorbance of each known
solution is measured and then a calibration curve of
concentration vs. absorbance is plotted.
The sample solution is fed into the instrument, and the
absorbance of the element in this solution is
measured. The unknown concentration of the element
is then calculated from the calibration curve.

Relation of AAS with UV-Visible spectroscopy
UV-Vis spectroscopy: is also similar to AAS in number of ways;

Have the similar basic principle which is promoting electrons from lower energy level to a higher energy
level.


Both techniques uses similar steps to interpret results.

Dissimilarities;

AAS – uses ‘visible’ part of the emission spectrum


UV-Vis – uses ‘ultraviolet’ part of the emission spectrum

Working of Atomic Absorption spectrometer:
In actual practice;
A meter is adjusted to zero absorbance. When a blank (unionized water) is sprayed into the flame
and unsaturated light of hollow cathode lamp is passes onto the read out device.
Next, when solution containing absorbing species is introduced, a part of light is absorbed, results in the
decrease light intensity falling on photomultiplier detector. And produce a deflection in meter needle.

Atomic absorption spectrum:

Spectrum of radiations shows a series of a dark lines in a continuous band.






Absorption bands:

Regions in spectrum from where radiations have been absorbed by the substance in sample.

Resonance spectral lines:

They are stable intense radiations appears a resonance spectral lines. They should be narrow as
compared to the width of absorption bands / lines.

Interference during AAS
The concentration of the analyte element is considered to be proportional to the ground state atom
population in the flame, any factor that affects the ground state atom population can be classified as an
interference.
Factors that may affect the ability of the instrument to read this parameter can also be classified as an
interference. The different interferences that are encountered in atomic absorption spectroscopy are;

Absorption of Source Radiation:

Element other than the element of interest may absorb the wavelength being used, and thus
interfere.

Ionization Interference:

The formation of ions rather than atoms causes’ lower absorption of radiation. This problem is
overcome by adding ionization suppressors or non-ionizing agents.
Example:
Certain, atoms like Na+
and K+
, they ionizes at low temperature therefore non-ionizing agent
CsCl2 is used.

Self-Absorption:

The atoms of the same kind that are absorbing radiation will absorb more at the center of the line
than at the wings and thus resulting in the change of shape of the line as well as its intensity.

Back ground Absorption of Source Radiation:

This is caused by the presence of a particle from incomplete atomization. This problem is
overcome by increasing the flame temperature.

Physical Interference:

Physical properties e.g. viscosity, surface tension, vapor pressure, and density, of the sample
should be similar to the standard.

Spectral interference:

It is caused by overlapping of any radiations with that of characteristic radiations of test element
to be estimated. This type of overlapping can be overcome by selecting other spectral lines.

Chemical interference:

Certain metals like calcium Ca+2
and magnesium Mg+2
make strong bonds with phosphate PO-3
as Ca3(PO4)2 and Mg3(PO4)2. Then in this case there will be no absorbance by Ca+2
and Mg+2
. To
overcome such problem;

By increasing the temperature so that bond dissociation occur easily.


By addition of releasing agent (e.g. LaO2)



By addition of chelating agent (e.g. EDTA)



Back ground correction:

When light spectra is released, monochromator (the wavelength selector) selects specific
wavelength of specific metal. But, sometimes same waves interfere with spectra.
Therefore to overcome this problem a blank is run in the instrument and calculated. And then it
is subtracted from the readings of the sample.
Advantages and disadvantages of Atomic Absorption Spectroscopy
The technique has it some merits and demerits discussed below;
Advantages:

Precise and accurate results can be obtained by the usage of this technique.


It is a very sensitive. It can detect concentrations as small as a few parts to g / Litre (parts per million)



It is generally very specific as the set wavelength is strongly absorbed by the particular metal ion being
analysed (and not by other components).


Only a little quantity of the sample is required about 1ml – 2ml.


It is a lifesaving technique. In japan from 1932 to 1968, AAS was used to identify the reason why
over 3,000 residents who lives near the Minimata Bay started showing neurogical problems and
pregnant women starts giving birth to impaired children. Scientist starts taking samples and
performing AAS process; AAS results shows a very high concentration of mercury in their blood.
This result on stopping the company, Chisso Corporation who dumped approximately 27 tons of
mercury in the bay.

Disadvantages:

It is a cost effective technique.


Flame atomic absorption spectroscopy, can analyze only liquid sample.



Graphite atomic absorption spectroscopy should not be used when ordinary flame AA would do as well,
since there are disadvantages relating to sample size and precision.


Applications of Atomic Absorption Spectroscopy
There are many applications for atomic absorption;
Clinical analysis:
By the use of this technique we can detect deficiencies / excessive amounts of certain metals in our
biological fluids such as; blood and urine.
Environmental analysis:
The technique is widely used for the monitoring of our environment. It is used to analyze metal ions that
are polluting the soil, air and water. And thus to find out the levels of various elements in rivers,
seawater, drinking water, air, and petrol.
Pharmaceuticals:
In some pharmaceutical manufacturing processes, minute quantities of a catalyst used in the process
(usually a metal) are sometimes present in the final product. Therefore, by using AAS the amount of
catalyst present can be determined.
For example in vitamin preparations.
Industry:

Raw material analysis: Many raw materials are examined and, AAS is widely used to check that the
major elements are present and that toxic impurities are lower than specified.

For example; in concrete, where calcium is a major constituent, the lead level should be low
because it is toxic.

Food industry: The technique is used for trace elements in food analysis. Where, it is used to track
harmful metals in our food/drinks.



Cosmetics industry: The technique is used for the trace element analysis of cosmetics in cosmetics
industry.

For example to analyze the specific allergic metal in cosmetics.

Petroleum industry: In petroleum industry the technique is used to analyze the metals present in
engine oils.

For example to check the presence of anti-knocking agent tetraethyl lead (TEL).
Mining:
By using AAS the amount of metals such as gold in rocks can be determined to see whether it is worth
mining the rocks to extract the gold.

Basic concepts
Emission spectrum:
The collection of spectral lines produced by an excited atom is called emission spectrum and will be
characteristic of that atom.
Principle:
When the element is heated in the flame, the absorption of energy by the ground state electron in an
atom results in excitation of some on these electrons to higher energy resulting in excitation.
A solution of sample to be analyzed is sprayed into flame possessing the thermal energy require to
excite the element at which it will radiate its characteristic bright line emission.
Types of emission spectrum
There are three types of emission spectrum;
1. Line emission spectrum
2. Band emission spectrum
3. Continuous emission spectrum
1. Line emission spectrum:
It is consist of sharply defined and often widely and irregularly spaced individual lines of a single
wavelength. These spectra are characteristic of element. They are also called atomic spectrum.
2. Band emission spectrum:
It consist of group of lines each of which has single wavelength that becomes closely spaced as they
approach the end of band. They are also called molecular spectrum.
3. Continuous spectrum:
They are obtained when solids are heated to incandescence. They are characterized by absence of any
sharp lines as a function of wavelength. On the other hand when gases and vapors are heated to high
temp they yield a series of bands or lines.

Atomic emission spectroscopy
Definition: When we study the emission of energy by
the atoms in the flame, we call this technique as Atomic
Emission Spectroscopy AES.
Atomic Emission Spectroscopy (AES) is an analytical
technique that measures the concentrations of elements
in samples. It makes use of the emission of light by these
elements, in order to measure their concentration.
Principle:
In atomic emission the sample is atomized and the analyte atoms are
excited to higher energy levels. The analyte concentration is
determined from the amount of emission.
The analyte atoms are promoted to a higher energy level by the
sufficient energy that is provided by the high temperature of the
atomization sources. The excited atoms decay back to lower levels by
emitting light. Emissions are passed through monochromators or
filters prior to detection by photomultiplier tubes.
The change in energy can be calculated as;
= −
=
=
So, the equation will be;
= 
Where;
 ΔE – Change in energy
 E2 – Excited energy
 E1 – ground state
 h – Planck’s constant
 c – velocity of light
  – wavelength
Concentration measurements are usually determined from a working curve after calibrating the
instrument with standards of known concentration.
The basic equation is; ∝

Instrumentation:
The instrumentation of atomic emission spectroscopy is the same as that of atomic absorption, but
without the presence of a radiation source. Atomic absorption spectrometer have three principal
components;
1. Atomizer (Flame, Graphite furnace, ICP)
2. A Monochromator
3. A detector, and read out device
Atomizer:
The major energy source in Atomic Emission spectroscopy are;
 Graphite furnace: This technique should be used only when the sample size is small and/ or
when a greater sensitivity is needed. The substance sample is dried at 200o
C for 60 sec. Then it
is burned at 1200o
C for 30 sec. to burn all organic sample. And then finally the atomization is
brought about by heating at 2700o
C for 60 sec.

 A flame: The flame (1700 o
C – 3150o
C) is most useful for elements with relatively low
excitation energies like sodium potassium and calcium.

 Inductively coupled plasma: The ICP (6000o
C – 8000o
C) has a very high temperature and is
useful for elements of high excitation energies.

 Electric arc: sample is heated by an electric arc.

 Electric spark: Sample is excited in high voltage spark.

Monochromator:
This is an important part in an Atomic Emission spectrometer. It is used to separate out all of the
thousands of lines. Without a good monochromator, detection limits are severely compromised.
A monochromator, in atomic emission sepectroscopy, is simply a wavelength selector. It is used to select
the specific wavelength of light which is emitted by the sample, and to exclude other wavelengths. The
selection of the specific emitted radiation allows the determination of the selected element in the
presence of others.
Detector:
The selected wavelength by the monochromator is directed onto a detector that is typically a
Photomultiplier tube, whose function is to convert the light signal into an electrical signal proportional
to the light intensity.
The processing of electrical signal is fulfilled by a signal amplifier. The signal could be displayed for
readout, or further fed into a data station for printout by the requested format.
Calibration curve:
A calibration curve is used to determine the unknown
concentration of an element in a solution. The
instrument is calibrated using several solutions of
known concentrations. The emission of each known
solution is measured and then a calibration curve of
concentration vs. emission is plotted.
The sample solution is fed into the instrument, and the
absorbance of the element in this solution is
measured. The unknown concentration of the element
is then calculated from the calibration curve

Comparison between Atomic Absorption and Emission Spectroscopy
Atomic Absorption spectroscopy Atomic Emission spectroscopy
Measure trace metal concentrations in complex Measure trace metal concentrations in complex
matrices. matrices.
Atomic absorption depends upon the number of Atomic Emission depends upon the number of
ground state atoms. excited atoms.
Sensitivity:
Both techniques are used to
detect metal or metalloids in
the sample.
The detectable elements by
this technique are in pink
colored in the periodic table;

Molecular
Fluorescence
Spectroscopy

Introduction
Luminescence:
It is defined as, the emission of photons from electronically excited state.
Luminescence is divided into two types, depending upon the nature of the ground and the excited states.
There are two states;
1. Singlet state
2. Triplet state
1. Singlet state
In a singlet excited state, the electron in the higher energy orbital has the opposite spin orientation as
the second electron in the lower orbital. These two electrons are said to be paired. Return to the ground
state from an excited singlet state does not require an electron to change its spin orientation.
2. Triplet state:
In a triplet state these electrons are unpaired, that is, their spins have the same orientation. A change in
spin orientation is needed for a triplet state to return to the singlet ground state. For example in case of
free radicles.

Types of Luminescence:
The luminescence is divided into two main types;
1. Photoluminescence
2. Chemiluminescence
1. Photoluminescence
The molecules are excited by the interaction with photons of radiation. It is further of two types;
 Fluorescence:
It is further of two types;

Prompt fluorescence: S1  S0 + h. The release of electromagnetic energy is
immediate or from the singlet state.


Delayed fluorescence: S0  S1 T1 S1  S0 + h. This results from two
intersystem crossings, first from the singlet to the triplet, then from the
triplet to the singlet.


 Phospholuminescence: S0  S1  T1 S0 + h
A delayed release of electromagnetic energy from the triplet state. The intensity of
phosphorescence is low as electron loses some part of its energy during intersystem
crossing (S1  T1).
2. Chemiluminescence:
The excitation energy is obtained from the chemical energy of reaction.
Time frame of Processes Time (sec)
S0  S1 10-19
sec.
Interconversion (Intermediate state  stable S1 state) 10-15
sec
S1  S0 10-10
sec.
Phosphorescence 10-3
– some seconds.

Fluorescence spectroscopy
Fluorescence spectroscopy FS is an analytical
technique that measures the concentration of
fluorescent substance present in the sample. It
makes the use of the fluorescence in order to
measure their concentration.
While, fluorescence is the emission of the light by a
substance which absorbs light or Electromagnetic
radiation.
Principle:
Molecular fluorescence spectrometry is based on the emission of light by molecules that have become
electronically excited subsequent to the absorption of electromagnetic radiation.
It uses the UV visible spectra along with some part of IR region of electromagnetic radiation. It is a
sensitive technique by which we can detect the sample raging from ppm – ppt.
Jablonski Diagram:
Let us understand this phenomenon from Jablonski
Diagram;
An electron was present in its ground state S0, it
absorbs light or electromagnetic radiation and gets
excited to a high intermediate energy level.
According to Kasha’s rule, the electron will require
to attain a stable energy level near to that
intermediate level, so it moves to S1.
After attaining a stable energy state it jumps back to
its ground state So either in non-radiative fashion, or
in radiative fashion thus, producing fluorescence.
The non-radiative fashion is due to the loss of energy during interconversion. In interconversion only
vibrational and rotational changes takes place within the molecules.
Properties of fluorescence:
Fluorescence is a specific property of each molecule and it depends upon the auxochrome of molecule.
The emitted light has two important characteristics;

It is usually of longer wavelength (lower energy) than the excited light. This is because part of the energy
associated with S state is lost as heat energy.


The emitted light is composed of many wavelengths which results in fluorescence spectrum.


Instrumentation
The description of various instrumental parts are given below;
1. Light source:
Two types of light sources are used;

Mercury-arc lamp: inorganic mercury lamps are used to energize the electron from ground state to
excited state. They provide steady and homogenous supply of light.


Xenon-Arc Lamp: Xenon gas is used as a source of light. It also gives steady supply. The wavelength
of light produce is about 300 – 1300 nm.

2. Excitation monochromator:
Various types of monochromators can be used to select the specific type of wavelength to fall on the
sample so that the sample excites that is why it is also called as excitation monochromator. It is also
called wavelength selector.
Commonly used monochromators are;

Prisms: various salt or glass prisms are used for the process.


Grating: In general, gratings are used in the design of the instrument and offer better resolution at
high frequency than the prisms. They offer much better resolution at low frequency.

3. Sample holder:
Cuvette or quarts cell is used as a sample holder. The cell is transparent from all sides so that the
fluorescence produced can be observed by the detector easily.
4. Monochromator:
A second monochromator is used to observe the produced fluorescence without difficulty. It acts as
wavelength filter. It is placed right angle to the first monochromator. It directs the fluorescence towards
the detector.
5. Detector:
The detector observes the fluorescence produce by the substance and generates the electrical signal. An
amplifier is attached that amplifies the intensity of the signal. The commonly used detectors are
phototube & photomultiplier tubes.
6. Read out devices:
The computer reads the detected signal and plots a graph.

Schematic diagram of Fluorescence spectrophotometer:
The schematic diagram of a simple fluorescence spectrophotometer is as fallows;
Applications of Fluorescence spectroscopy
Though the Fluorescence is a specific property of substances, but this type of spectroscopy has limited
applications as not all substances show fluorescence.
Some of its application are mentioned below;
1. Quinine was the first medicine that showed fluorescence.
2. The technique is used for the quantitative analysis as well as for qualitative analysis.
3. The technique is also useful for protein identification. This is done by tagging a fluorescent
molecule within the protein and fluorescence is observed.
4. This technique is used for the assay of different medications that shows fluorescence.
For example;
Medication Absorption wavelength Emission wavelength
λabsorption λemission
Amphotericin B 340 427
Diphenhydramine 305 412
Flurazepam 375 475
Reserpine 390 510

Factors affecting Fluorescence
The various factors affecting fluorescence are discussed below;
1. Temperature:
Increase in temperature will lead to the decrease in intensity of fluorescence. It happens because by
increasing the temperature the collision between fluorescent and other molecules and it loses its energy.
2. Oxygen:
Presence of oxygen leads to the oxidation of substance and thus causes decreases the intensity of
fluorescence produced.
3. pH:
Fluorescence of various chemicals depends upon different pH values. Most of the compounds give
fluorescence on neutral pH and some on alkaline pH. For example; Aniline gives fluorescence at pH &
(neutral.)
4. Quenching:
There are some molecules which are called quenching molecule. These molecules decreases the
fluorescence either by collision or by any other way like by changing the viscosity etc.
5. Rigid Planer structure:
The fluorescence produced by a rigid molecule is better as compared to the fluorescence produced by a
non-rigid structure.
For example; the fluorescence of Flourene is good as compared to the fluorescence produced by Biphenyl.
6. Electron withdrawing group:
The fluorescence is due to the π- electrons, thus the presence of an electron withdrawing group will lead
to the decrease in fluorescence of the substance as it draw these π- electrons towards it.
For example; halogens X, Carboxylic group –COOH, –NO2 etc.
7. Electron donating group:
Similarly, the presence of an electron donating group will result in increase in the intensity of fluorescence.
As it donates electrons.
For example; methyl group –CH3, amino group –NH2, hydroxyl group –OH etc.

Infrared spectroscopy
It deals with the region of electromagnetic spectrum that lies in
the infrared region (frequency range 500cm-1
– 670cm-1
)
IR radiations: Electromagnetic radiations that are lower in
energy than visible radiations are called infrared radiations.
Absorption in IR region is due to the rotational and vibrational
levels. When radiations of frequency range less than 100 cm-1
are absorbed, molecular rotation takes place in the substance.
Therefore, on the bases of molecular rotation, the vibrational
spectra appears as vibrational-rotational bands.
When IR light is passed through the sample the vibrational and rotational energies of the molecules are
increased. There are two types of vibrations;
1. Stretching vibration:
In this type of vibration the distance between the two atoms increases or decreases but the atoms
remains at the same bond axis. There are two types of stretching vibration;

Symmetric stretching: in this type the moment
of atoms with respect to the particular atoms
with respect to particular atom in the same
molecule is in the same direction.


Asymmetric stretching: in case of asymmetric
stretching one atom approaches the central atom
while the other departs from it.

2. Bending vibration:
In this type of vibration the position of atoms changes with respect to the original bond axis. We can say
that stretching absorption of the bond appears at higher frequencies, (high energy) as compared to the
bending absorption of the same bond. There are four types of bending vibrations;

Scissoring: in this type two atoms approaches each other.


Rocking: in this type the movement of atoms takes place in the same direction.



Wagging: in this type two atoms moves up and below the plane with respect to the central atom.



Twisting: in this type one of the atom moves up the plane while the other moves down the plane with
respect to central atom.


Introduction to IR Spectrometer:
The following figure gives the schematic representation or block diagram for the use of
spectrophotometer.
In this spectrometer the source of radiant energy is “Nernst glower.” It is consist of Zirconium and
Yttrium oxides in the shape of tube which is electrically heated to 1500o
C to 2000o
C because IR rays are
not transmitted by glass.
A prism made of salt (NaCl) is used as a monochromator. The radiations from the Nernst glower are
polychromatic. When these are passed through the salt prism, the different wavelength got separated a
slit is placed in the path of radiation emerging from the prism so that only radiant energy of the desired
wavelength passed through and falls onto the solution under examination.
The radiant energy transmitted by the solution is then allowed to fall on a detector for measuring the
intensity of transmitted infrared radiations.
Thus, IR spectrometer can give the absorption spectrum of a substance. Therefore, by analysis the
spectrum and the information regarding the structure of the substance can be obtained.
Regions of IR:
There are three main regions of IR;
Near IR IR region Far IR
0.8 mµ to 2.5 mµ 2.5 mµ to 15 mµ 75 mµ to 200 mµ
Absorption in IR region is mainly due to the changes in the rotational and vibrational levels of molecules.

Determination of IR spectrum of a substance
There are two method of determining the IR of the solid compound or substance;
1. MULL method
2. KBr disc method
MULL method: (Paraffin commonly called NUJOL)
1. Take 15g to 20g of sample in a clean pestle mortar and powdered it thoroughly.
2. Now add to it 2 drops of purified paraffin and continue the trituration until very smooth paste of
uniform consistency is achieved.
3. Now transfer the slurry to NaCl window (tablet/ window grooved)
4. Now place the other window onto the cell part and finally switch on IR machine to get various
spectrum of the sample.
KBr method:
1. Take the 100mg of the dried KBr in a clean pestle and mortar and grind it thoroughly with 1mg
of sample.
2. Now carefully place the sample mixture into processing chamber of the mold (dye). In such a
manner that it is held between the polished surface of the bottom and top processing dyes.
3. Subsequently, attach the chamber to the vacuum line and switch on the vacuum pump.
4. Initially applied a slight negative pressure to as to compact the powder and then gradually
increasing the pressure less than 15mm Hg for 30 seconds. Finally enhance the pressing force to
10,000 pound/ inch2
pressure for 1 to 2 minutes.
5. Now release the pressure and remove the dyes and take out the disc from the mold and kept it
into a position onto the sample holder to study the various spectrum of organic compounds.

Interpretation of IR:
IR spectrum is divided into two parts;

Functional group region

In case of functional group region the range is 4000 cm-1
– 1600 cm-1
.

Finger print region

In case of finger print region the range is 1600 cm-1
– 625 cm-1
.
As, the recent approach is to examine the functional group region because most the compounds
have only few strong bonds due to characteristic stretching vibrations of their functional group.
Example:

The functional group such as C-H, O-H, and C-N absorbs in the region of 3700 cm-1
– 2500 cm-1
due to
their stretching vibrations.


The absorption due to triple bond C≡C, C≡N occurs in the region of 2300 cm-1
– 2100 cm-1
.


The absorption due to double bond that is C=C, C=O occurs in the region of 1900 cm-1
–1600 cm-1
.

The confirmation of functional group should also be checked in the other region of spectrums as well.
Example: An aliphatic acetate absorbs not only at 1740 cm-1
but also at 1240 cm-1
.
The characteristic bands corresponding to the aromatic rings falls in the region of 1600 cm-1
– 1450 cm-1
but aromatic compounds also shows absorption bands in the region of 900 cm-1
– 700 cm-1
.
The finger print region the range is 1600 cm-1
– 625 cm-
1. It provides a set of absorption bands of each
compound and serve as finger print region.
The structural information is derived from the presence of or the absence of characteristic absorption
bands of various functional groups.

Instrumentation of IR Spectrophotometer:
The IR spectrophotometer are based on either single monochromator or double mnochromator. The
important features of an IR spectrometer are as follows;
1. IR source
2. Monochromator
3. Detector
4. Mode of operation
1. IR Source:
The most important IR sources are electrically heated rods of the following types;
 Sintered mixtures of the oxides of zirconium (Zr) & yttrium (Y), also known as Nernst glower.
 Silicon carbide (glow bar)
 Various ceramic material (clay)
2. Monochromator:
Three types of substances are normally used as monochromator;
 Metal Halide prisms: various metal halide prism such as Pot. Bromide KBr and Lithium
Fluoride LiF have been used.
 NaCl prism: Sod. Chloride can be used for the whole of the region from 4000cm-1
– 650cm-1

 Grating: in general, gratings are used in the design of the instrument and offer better resolution at
high frequency than the prisms. They offer much better resolution at low frequency.
3. Detectors:
There are three types of detectors used in IR region. They are discussed below;

Thermocouple: (thermophiles) if dissimilar metals used are jointed head to tail then a
difference in temperature between head and tail causes a current flow in wires. This current
shall be directly proportional to the intensity of radiation falling on thermocouple and hence
they are used in the IR region.



Golay detector: in this detector the absorption of IR radiations affords expansion of an inert gas in a
cell chamber the current from the photocell is directly proportional to the incident radiations.



Bolometers: they are related with increase in resistance of a metal with increase in
temperature. For example; when two platinum foil are appropriately incorporated into a
weightstone bridge and radiations is allowed to fall on the foil and a change in resistance is
noted. Just like thermocouple the bolometers are used in IR region.


4. Mode of operation:
The following steps are involved for the operation of the IR instrument;
1. The light from IR source [A] is splitting equally into two beams one of which [B] is sample beam
is passed through the sample i.e. the sample beam while the other serves as a reference beam.
2. These two beams are reflected on a rotated segmented mirror called Chopper [C] which helps to
pass the sample and reference beam to the monochromator grating [D].
3. The grating rotates slowly and transmits individual frequencies to the detector thermophile [E]
which converts IR energy to the electrical energy.
4. When a sample has absorbed a certain quantum of light of specific frequency. The detector shall
be receiving alternately from the chopper an intense beam (reference beam) and relatively
weak beam (sample beam). It will generate alternating current (AC Current) flowing from the
detector to the amplifier [F].
5. This out of balance signal received by the amplifier is coupled to a small servomotor [G]. That
gives an optical wedge [H] in the reference beam until the detector receives light of equal
intensity from sample and reference beam.
6. The slightest movement of the wedge (attenuator) is further coupled to an ink pen recorder [J]
and finely gives the various absorption bands (signals) on a printed chart.
Schematic diagram of IR Spectrophotometer:

Applications of IR Spectrophotometer
The important applications of IR spectroscopy are discussed below;
1. Qualitative analysis:
We can identify the functional group of many organic compounds and drugs. For example; benzoic acid,
salicylic acid, phenol, aniline, P-aminophenol.
We can identify the functional group as well as aromatic ring of many organic compounds by IR
spectroscopy. For example; acetanilide, Paracetamol, Aspirin etc.
2. Quantitative analysis:
By using IR spectrum of the sample of drug and by making the comparison of IR spectrum of standard
drug we can also determine the percentage purity of the drugs.
3. Hydrogen bonding:
By using IR spectroscopy we can determine the H-bonding of the organic molecules highly
electronegative atoms such as Nitrogen, Oxygen and Fluorine are involved in strong H-bond formation.
4. Purity of sample:
Generally the pure sample shows fairly sharp spectrum whereas the material of high molecular weight
generally shows poor spectrum because of the presence of several kinds of functional groups.
5. Chromatographic separation studies:
The process of chromatographic separation can readily be monitored by taking the spetra of selected
fractions.
6. Determination of aromaticity:
By taking the IR spectrum of different organic aromatic drugs we can study the relative proportion of
saturated and unsaturated rings present in hydrocarbons.
7. Tatumerism:
Tatumeric equilibria can be studied with the help of IR spectroscopy. Most common system such as
keto-enol and Lactum to Lactum contain a group such as C=O, OH, NH2, C=S group which shows
characteristic frequency which make it possible for identification of particular drug.
8. Identification of different groups and bonds:
By taking the IR spectrum of different functional groups we can identify on the bases of spectrum having
their specific frequency range.

Example:
Group Bond Frequency range cm-1
Alkyl C-H 2853 – 2962
Alcohol O-H 3590 – 3650
Amine N-H 3300 – 3500
Similarly in case of bond and frequency;
Bond Frequency range cm-1
C=C 2100 – 2260
C=O 2220 – 2260
C≡C 1620 – 1680
C≡N 1630 – 1780
7. Organic reactions: We can study the IR spectrum of organic reactions.
8. Dipole moment: We can study the dipole moments of organic compound.
9. Dissociation constant: We can study the degree of dissociation constant of organic compounds.
10. Complex formation: We can determine complex formation study of reactions.
11. Finger prints: We can study the finger print region of organic compounds.
Some of the important examples of raw material and dosage form are as follows;
Raw material;

Paracetamol raw material


Aspirin raw material



Ibuprofen raw material



Chlorocaine raw material



Chloroquinie raw material

Dosage forms;

Aspirin tablet


Paracetamol Tablet and syrup



Ibuprofen tablet and syrup



Ponston tablet (Mefanamic acid)



Neubrol tablet (orphendrine + paracetamol)


Nuclear Magnetic Resonance NMR
Nuclear magnetic resonance spectroscopy is a powerful analytical technique used to
characterize organic molecules by identifying carbon-hydrogen frameworks within molecules.
It can be defined as, “It is the study of absorption of electromagnetic radiations by atomic nuclei in the
radiofrequency region in the presence of magnetic field is called NMR spectroscopy.”
Principle:
The absorption of electromagnetic radiation in radiofrequency region by the nucleus results in
the change in orientation of spinning nucleus in an applied magnetic field.
Types of NMR:
Two common types of NMR spectroscopy are used to characterize organic structure;
 1
H NMR is used to determine the type and number of H atoms in a molecule.


 13
C NMR is used to determine the type of carbon atoms in the molecule.

Basic theory:
A. Spinning nucleus:
When a charged particle such as a proton spins around its axis, it creates a magnetic field. Thus, the
nucleus behaves as a tiny bar magnet.
B. Behavior under applied magnetic field:
Normally, these tiny bar magnets are randomly oriented in space. However, in the presence of an
external magnetic field B0, they are oriented with or against this applied field. More nuclei are oriented
with the applied field because this arrangement is lower in energy.
The energy difference between these two states is very small (<0.1 cal).

C. Spin flipping:
In a magnetic field, there are now two energy states for a proton;
1. α-spin state: A lower energy state with the nucleus aligned in the same direction as B0
2. β-spin state: A higher energy state in which the nucleus aligned against B0.
Transition of a proton from α-spin state to β-spin state by the absorption of radiofrequency of
electromagnetic radiation is referred to as “spin flipping.”
The energy difference between these two nuclear spin states corresponds to the low frequency RF
region of the electromagnetic spectrum.
D. NMR signals:
Thus, two variables characterize NMR;

An applied magnetic field B0, the strength of which is measured in tesla (T)



The frequency of radiation used for resonance, measured in hertz (Hz), or megahertz (MHz)

Both these variables are proportionally related;
Therefore, the NMR signals can occur by keeping one of both constant and changing the second.
i. Field sweep (ν is constant & B0 is varied)
ii. Frequency sweep (ν is varied & B0 is constant)
Note: Only nuclei that contain odd mass numbers (such as 1
H, 13
C, 19
F and 31
P) or odd atomic
numbers (such as 1H and 7N) give rise to NMR signals.

NMR spectrum:
Modern NMR spectrometers use a constant magnetic field strength B0, and then a narrow range of
frequencies is applied to achieve the resonance of all protons. Protons in different environments absorb
at slightly different frequencies, so they are distinguishable by NMR.
An NMR spectrum can be defined as, “it is a plot of the intensity of a peak against its chemical shift,
measured in parts per million (ppm).”
NMR absorptions generally appear as sharp peaks. The increasing chemical shift is plotted from left
to right. Most of the protons absorb between 0-10 ppm.
The terms “upfield” and “downfield” describe the relative location of peaks.

Upfield means to the right.


Downfield means to the left.

NMR absorptions are measured relative to the position of a reference peak at 0 ppm on the δ-scale due
to tetramethylsilane (TMS).
Following features of a 1
H NMR spectrum provide information about a compound’s structure:
a. Number of protons
b. Position of protons (chemical shift)
c. Intensity of signals
d. Spin-spin splitting

a. No. of protons:
The number of NMR signals equals the number of different types of protons in a compound.

The equivalent protons give the same NMR signal.



Protons in different environments give different NMR signals.


To determine equivalent protons in cycloalkanes and alkenes, always draw all bonds to hydrogen.


In comparing two H atoms on a ring or double bond, two protons are equivalent only if they are
Cis (or Trans) to the same groups.



Proton equivalency in cycloalkanes can be determined similarly.


b. Positions of protons or Chemical shift:
The shift in absorption position of NMR which arises due to the shielding or deshielding of protons by
electrons is called chemical shift.

Chemical shift to the Right:

The proton is a molecule that is surrounded by a cloud of electrons. When magnetic field is applied it
induces the electron to circulate around the nucleus perpendicular to the applied magnetic field.
These circulating electrons produces their own magnetic field in a direction opposite to the applied
magnetic field. Since, the induced magnetic field of electrons is in opposite direction to the applied
magnetic field. The effective magnetic field experiences by the nucleus is reduced.
( ) = ( ) − ( )
Thus, the circulating electron can partially shield the nucleus from the applied magnetic field.
The nucleus is said to be shielded and such type of shielding is called Diamagnetic shielding.
Since, the nucleus is shielded therefore, it needs a lower frequency to achieve resonance. Lower
frequency is to the right in an NMR spectrum, toward a lower chemical shift, so shielding shifts
the absorption upfield.

Chemical shift to the Left

The less shielded the nucleus experiences the more of the effective magnetic field (B0) it.
Thus, the deshielded nucleus experiences a higher magnetic field (B0) strength. Therefore, it needs a
higher frequency to achieve resonance. Higher frequency is to the left in an NMR spectrum, toward
higher chemical shift. So, deshielding shifts an absorption downfield. Protons near electronegative
atoms are deshielded, so they absorb downfield.
The effective field may be different for different nucleus because of shielding effect. Thus
their resonance energy will differ slightly.

Factors affecting Chemical shift
Following are some factors which affect the chemical shift;
1. Electronegativity:
The extent of shielding depends upon the electron density or electronic cloud around the nucleus and
electrons depends upon the electronegativity. As the electronegativity increases, the electrons will be
drawn away from the nucleus and shielding will be decreases. Thus, the nucleus will be deshielded in the
case and experiences more field. So, deshielding shifts an absorption downfield.
2. H-bonding:
Increase in H-bonding will lead to increase in deshielding. So, deshielding shifts an absorption downfield.
3. Solvent effect:
Solvent variation results in dramatic change in chemical shift. For example;

Aromatic solvents (benzene) cause deshielding.


Acidic solvents causes deshielding.



Non-aromatic solvents causes shielding.


4. Magnetic anisotropic effect:
It is related to the geometry. A proton may experiences additional shielding or deshielding
depending upon its orientation relative to the induced magnetic field caused by the electronic
circulation such effect is called magnetic anisotropic effect.
Example 1:
If the induced magnetic field is in the direction of applied magnetic field then these both fields will
reinforce each other. The protons thus feel a stronger magnetic field and a higher frequency is needed
for resonance. Thus they are deshielded and absorb downfield. In case of Benzene;

Example 2:
In some cases the induced magnetic field is in opposite direction to the applied magnetic field. Thus, the
proton feels a weaker magnetic field, so a lower frequency is needed for resonance. The nucleus is
shielded and the absorption is upfield.
5. Addition of Lanthanide shift reagent:
They are complexes of lanthanides metal when we add reagent they alter chemical shift of
protonby forming complexes. They are used to simplify the NMR spectrum.
Formula:
= ( ) − ( ) =

ν(sample) = Frequency of sample



ν(TMS) = Frequency of TMS



ν0 = Frequency of instrument

The frequency of sample and TMS is in Hertz (Hz). While, the frequency of the instrument ν0 is in Mega
Hertz (MHz). That’s why we take it in parts per million (ppm).
= = =

c. Spin-spin splitting or Spin-spin coupling:
“Splitting of resonance bands in NMR into doublets, triplets, quartets etc. is called as spin-spin splitting.”
Or
“The phenomenon that describes magnetic interaction between neighboring non-equivalent nuclei in
NMR.”
Peaks are often split into multiple peaks due to magnetic interactions between nonequivalent protons
on adjacent carbons atoms. The splitting of absorption signal is a set of peaks. It is a multiplet (2 =
doublet, 3 = triplet, 4 = quartet, 5=pentet, 6=hextet, 7=heptet…..)
Mechanism of Splitting or Coupling:
Let’s understand this with the example of Tri bromoethane.
When it is placed in an applied magnetic field (B0), the adjacent protons Ha and Hb can each be aligned
with () or against () B0.
Thus, the absorbing proton feels three slightly different magnetic fields;
1. One slightly larger than B0 (ab)
2. One slightly smaller than B0 (ab)
3. One the same strength as B0 (ab)
As, the absorbing proton feels three different magnetic fields, it absorbs at three different frequencies in
the NMR spectrum, thus splitting a single absorption into a triplet because there are two different ways
to align one proton with B0, and one proton against B0— i.e. ab and ab—the middle peak of the
triplet is twice as intense as the two outer peaks, making the ratio of the areas under the three peaks
1:2:1.
Thus, two adjacent protons split an NMR signal into a triplet. When two protons split each other, they
are said to be coupled.
Significance of coupling:
1. It helps in identifying types of protons.
2. It is helpful in identifying no. of protons of each type.
3. It is a helping tool in identifying no. of neighboring protons.

Examples:
Coupling constant:
It is defined as, “the distance between the centers of the adjacent peaks in a multiplet is usually a
constant, and it is known as coupling constant. It is represented by ‘J’. It varies from compound
to compound.
It is expressed in Hertz or Cycles/ sec. It is a constant that depends upon the structural relationship
between the coupled protons. It is independent of the applied field and the nature of solvent.
Significance:

It is useful to distinguish between singlet and doublet.



It also helps to differentiate between 1 quartet and 2 doublets.


Rules of Splitting
1. The splitting is into one more peak than the number of H’s on the adjacent carbon(s), This is the
“n+1 rule”
2. The relative intensities are in proportion of a binomial distribution given by Pascal’s Triangle
1 singlet
1 1 doublet
1 2 1 triplet
1 3 3 1 quartet
1 4 6 4 1 pentet
1 5 10 10 5 1 hextet
16 15 20 15 6 1 heptet
3. Equivalent protons do not split each other.
4. Protons that are farther than two carbon atoms apart do not split each other

5. If Ha and Hb are not equivalent, splitting is observed when;
6. Splitting is not generally observed between protons separated by more than three  bonds.
7. Complex splitting: If there is more than 2 interacting groups, the bond multiplicity of A as spit by
B & C is expressed as;
(nB+1) (nC+1) = No. of lines

Instrumentation and Data Analysis
The NMR instrument consist of following parts;
1. Sample tube
2. Magnet
3. Field sweep coil
4. Radiofrequency transmitter or Generator
5. Radiofrequency receiver
6. Amplifier & Readout device
1. Sample tube:
The sample holder in NMR is normally tube-shaped
and is therefore called the sample tube. It is a tube of
15cm length and 5mm diameter.
It is made up of borosilicate glass. It is inert, durable
and transparent to radiofrequency radiations. Glass
or Pyrex tubes are commonly used. These are
sturdy, practical, and cheap.

2. Magnet:
The magnet in NMR spectrometer must be strong, stable, and produce a homogeneous field.
Homogeneous in this context means that the field does not vary in strength or direction from point to
point over the space occupied by the sample.
The magnet is so large that this instrument comes with its own staircase so that the analyst can insert
and remove the sample tube from the top of the probe.
Three types of magnates can be employed in NMR spectroscopy;
a. Permanent magnates
b. Electromagnets
c. Super conducting magnets
a. Permanent:
It is Cheap and it is convenient to use, but it not frequently used as it lacks flexibility and
no variations can be made.
b. Electromagnets:
The type of magnet is insensitive to temperature. The advantage of this magnet is that;
 The flux density or the strength of applied magnetic field can be controlled.
 The current passing through the coil can be controlled.
c. Super conducting magnets:
They are most powerful magnets as compared to the rest. They possess high resolution. All
NMR spectrometers having frequency above than 100 MHz are based on Helium cooled
super conducting material.
3. Field sweep coil:
It is a coil either wrapped around or placed between the two poles of the magnets. It is used to stable
the applied magnetic field, it also homogenize the frequency of magnets.
The strength of applied magnetic field can be varied by varying the passing current in the sweep coil.
4. Radiofrequency generator:
It transmits the radiations of controlled frequency to the sample in the direction to the magnetic field.
The radiations are applied in such a way that they are right angle to radiofrequency receiver. This
provide signals require to induce transitions.
5. Radiofrequency receiver:

It consist of a receiver coil. It is coiled around the sample tube at right angle to the applied field as
well as transmitter coil.
6. Amplifier & readout device:
Signals from the receiver coil is weak, therefore they are amplified and recorded mechanically, or pre-
calibrated charts with respect to reference compounds.
7. Schematic diagram of NMR:

Reference standard for NMR:
The position of absorption of various protons cannot be determined separately with accuracy. A
standard reference is used either an internal reference or external reference. Therefore, the NMR
absorptions are measured relative to the position of a reference peak at 0 ppm on the delta scale.
Reference is assumed to be free of shielding effect and it requires less magnetic field. The standards
used for NMR must be chemically inert, miscible with most organic compounds and must have low
melting point.
The most commonly used reference standards in NMR are;
1. TMS – Tetramethylsilane, is a volatile inert compound that gives a single peak upfield. Its boiling
point of is 27o
C. It is generally used NMR standard for organic compounds.
2. DSS – Dimethyl silapentane sulfonic acid, is more often used in protein experiments in water.
Solvents used in NMR
The solvents which are mostly used for the NMR spectroscopic analysis are;

Carbon tetra chloride CCl4


Carbon disulphide CS2



Deutrated Chloroform CDCl3



Deurated Benzene C6D6



Deutrated water or Heavy water D2O

Properties of a good solvent:
The solvent used for NMR must have following properties;

It should be cheap and easily available.


It should be non-viscous.



It should be chemically inert to the sample and sample holder.



It should dissolve maximum amount of organic compound.



The solvent must devoid of its own protons.



Magnetic anisotropy.


Working of NMR spectrometer:
The working of NMR spectrophotometer consist of following steps;
1. 0.5 ml of 15% sample solution in test tube is made. Then add few drops of reference standard
DSS or TMS.
2. The Sample tube is then placed between poles of a magnets.
3. Radiofrequency radiations is made to fall on sample by radiofrequency generator.
4. The field strength is increased by increasing the current in sweep coil. As a result precessional
frequency of each set of protons increase until resonance with radiofrequency source take place.
5. Now the detector produces a signal.
6. Signal from the detector is amplified and then recorded.
Uses of NMR spectrometer:
1. It is used in research and development.
2. It is used in biology of biological studies. For example; to check the structure of proteins.
3. It is used in food industry for moisture analysis.
4. It is used in agriculture.
5. It is used in pharmaceutical analysis and industry.

Applications of NMR
Spectrometer 1. Investigation of dynamic properties:
NMR can be used to investigate the dynamic properties of molecules such as;

H-bonding


Molecular information


Conformational isomer


Restricted rotation



2. Determination of optical activity:
Diastereomers differs in their NMR properties. So, this technique can be used for the determination
the optical activity.
3. Keto-enol tautomerism:
It is a more powerful analytical technique for quantitative and qualitative investigation of Keto-enol
equilibria. For example; keto-enol forms of Acetones.
4. Structure elucidation:
The structure of an unknown compound can be determined from;

No. of signals


Chemical shift (it indicates the type of electronic environment of the peotons)


Spin-spin coupling


5. Quantitative analysis:
The technique has been used to determine the molar ratio of compound in a mixture.
Percentage of each component = area under peak component/ total area under peak X hundred
6. Elemental analysis:
To determine the given type of magnetic nucleus in the sample.
7. Drug macromolecular interaction:
We can study miscell formation of drug in the sample and macromolecule interaction.
8. Study of isotops:
Several nuclei in addition to protons that have magnetic movements can be studied y magnetic
resonance technique. For example; fluorine, phosphorous etc.
9. Moisture analysis:
Water absorbed in biological materials such as food products appear in NMR spectra as relatively sharp
bands.
10. Hydrogen analysis:
Percentage of hydrogen in an unknown sample may be determined easily and rapidly.

11. Cis-Tans isomers:
NMR can be used to distinguish between Cis-trans isomers.
12. Compound identification:
It can be used for compound identification that is, to confirm a compound in the sample.
13. NMR can also be used to confirm the Purity of a chemical compound.
14. NMR is used for the determination of surfactant chain length.
15. NMR is utilized to find the Iodine value of triglycerides

Basic concepts
Witt theory:
German scientist O.M. Witt in 1876 suggested the chromophore auxochrome theory for colored organic
compounds. The various terms which are used as fallows;
Chromophore: The graphs which has unsaturation and electron withdrawing effect have an
appreciable effect on the absorption of light and well present in conjugation are responsible for the
color of compounds (due to absorption in visible region) such groups are known as chromophore.
Chromogen: The compounds containing chromophore groups are known as chromogens. For example
N=O, -N=N, >C=S, >C=O, >C=C< etc.
Auxochrome: These groups are not responsible for colors but when present along chromophore groups are
responsible for deepening of the color. They are electron donating groups. For example -NH2, -OH.
Quinonoid theory:
According to this theory ortho and para quinonoid structures in a compound are responsible for its color.
Modern theory:
This theory is basically the modification of Witt & Quinonoid theory. It explains the colors of organic
compounds based on resonance effect and its correlation with absorption of light.
The light waves in UV and visible region have high energy. When light falls on a compound it gets
absorbed and results in three types of excitation in the molecule i.e. electronic, vibrational, rotational. If
E1 is the energy in ground stand and E2 is the energy in the excited state than ΔE energy required for
excitation will be;
ΔE = E2 – E1 =hν
The chromophore and auxochrome groups present in a compound causes deepening of color by
increasing the number of charged contributing structures during resonance effect.
The increased conjugation (delocalization) in a system shifts the absorption towards longer wavelength
region lower energy and is known as “Bathochromic shift.” Similarly shifting of the absorption towards
the shorter wavelength region (high energy) is known as “Hypsochromic shift.”
Example: Nitrobenzene is yellow in color due to presence
of nitro group. Whereas, para-nitroaniline is orange in
color. The nitro group is chromophore which produces
yellow color due to resonance effect. On the other hand
amino group is auxochrome which causes deepening of
color by increasing the no. of contributing structures
because of the presence of three nitro group.

UV-Visible Spectroscopy
Introduction:
There are two regions;

UV region 200nm to 400nm


Visible region 400nm to 850nm

When a beam of sun light is passed through a
prism it splits into seven colors i.e. red,
orange, yellow, green, blue, indigo, and
violet. This set of colors obtained by splitting
the white light is called spectrum. Different
colors are associated with different energy
and wavelength. For example red light has
smallest energy and longest wavelength.
Energy goes on increasing and wavelength goes on decreasing as we move from red to violet color.
Therefore, the violet color has the maximum energy and minimum wavelength.
Electromagnetic spectrum:
In addition to white light with its seven different colors or radiations, there are many more different
types of radiations. Some of them are more energetic and some are less energetic than the visible white
light. The electromagnetic spectrum consist of the following radiations;
(i) Cosmic rays: these rays come from sun and are known to be the radiations of highest
energy. They have wavelength less than 10-3
nm.
(ii) γ rays and X rays: they are less energetic than cosmic rays their wavelength range is
between 10-3
nm to 10-1
nm.
(iii) UV light: these are the rays which are less energetic but more energetic than visible light.
The range of wavelength of UV region is 200nm to 400nm.
(iv) Visible light: it is ordinary light and it is composed of several different radiations (from red
to violet) and the wavelength range is 400nm to 850nm.

Principle:
When a beam of electromagnetic radiation is passed through a compound certain radiations are
observed. There is some energy associated with energy radiation and is given by the following equation;
=
Where;
 ν is the frequency of radiations
 h is the Plank’s constant
The energy absorbed by the substance produces some changes within the molecule if the energy
absorbed is high it causes electronic excitation i.e. electrons are excited to higher level. The graph
between the amount of radiation absorbed by the sample and the wavelength of the radiation is called
absorption spectrum. It consist of bands with peaks of maximum intensity. The overall range of
electronic spectroscopy is from 180nm to 850nm.
Range of determination:
It records the spectra of compounds or drug molecules in the range of 180nm – 185nm. This range
consist of two parts;
 180nm – 400nm, which is range of UV radiation.
 400nm – 850nm, which is range of visible light
The spectra below 180nm cannot be taken because oxygen present in spectrophotometer absorbs these
radiations to record the spectra of substances below 180nm. Special vacuum conditions are needed.

Instrumentation and working:
The UV-visible spectrometer consist of following parts;
1. Light source
2. Monochromator (Prism type, Grating type)
3. Quarts cell
4. Detector (phototubes, photomultiplier tubes)
5. Recorder
The following figure gives the schematic representation or block diagram for the use of
spectrophotometer.
1. The solvent used to dissolve the sample are the ethanol, methanol, n-hexane and distilled
water. For recording the UV-visible spectrum the sample is dissolved in a solvent, which itself
don’t absorb light in that’s range.
2. A quartz cell made up of special glass of 1cm path length is used as a container for the sample
solution (sample holder).
3. The solution is exposed to UV-Visible light by the prism selector. The prism selector is rotating
continuously to emit lights of different wavelength.

Hydrogen lamp is used for emitting UV light.


Tungsten lamp is used for emitting visible light.


4. The instrument provides a graph between wavelengths of radiations absorbed and the intensity
of absorption.
5. The wavelength corresponding to top of peak shows the maximum absorption. The wavelength
is denoted by λ and maximum absorption is denoted by λmax
6. The intensity of absorption corresponding to the wavelength is called ‘Molar extinction
constant,’ and it is denoted by epsilon or Ɛ.

Beer’s Lambart’s Law
Beer’s Law:
When a beam of monochromatic light is passed through a substance dissolved in a non-absorbing
medium the absorption of light is directly proportional to the molar concentration of the substances.
Mathematically;
log10 ∝
This is referred as Beer’s law.
Lambart’s Law:
When a beam of light is passed through substance the absorption of light is directly proportional to the
path length of the substance.
log10 ∝
This is referred as Lambart’s law.
The two laws are combined to obtain the absorption of light by a substance therefore;
log10 ∝ .
log10 = Ɛ
Where;

Io stands for intensity of incident light


I stands for transmitted light



C stands for ‘concentration of substance in centimeter



Ɛ stands for proportionality constant known as molar extinction constant.

Expression of absorbance is;
log10 = ( )
So, final equation can be written as;
= Ɛ

Spectrophotometric determination of absorbance of
known solution;
Suppose we want to determine the amount of substances in the solution by spectrophotometric
method than the procedure will be as fallows;
1. Place the solution in a quartz cell.
2. Now put the cell in a cell holder and move it into a position so that the solution comes in a light
path.
3. Go on changing the wave length or the incident radiation and note the absorbance for each
value of the wave length.
4. Now plot a graph between absorbance and wavelength and find out the value of λmax
5. This wavelength is used in the measurement of absorbance throughout the experiment.
6. Prepare a series of different concentration of solution that is for known concentration measure
the absorbance of each known concentration at that wavelength as mentioned in step 2.
7. Plot a graph between different values of absorbance and concentration. This is called a
calibration graph or standard curve.

Applications of UV-Visible spectroscopy
Applications of UV-Visible spectroscopy are as fallows;
1. Detection of conjugation: With the help of UV-visible spectroscopy figure out the presence
of double and triple bond in a compound. The conjugation can be C=O, C=C, C≡C. the absorption
of that compound and by the observation of λmax values, we can also predict the location of
substituents.
2. Detection of functional group: it is possible to detect certain functional group with the help
of UV-Visible spectroscopy. Absence of absorption above 200nm is indication of the absence of
the conjugation.
3. Detection of geometrical isomerism (Cis/Trans): when compounds shows geometrical
isomerism. The Trans isomer shows the absorption at higher wavelength with larger values of
extinction co-efficient. That is absorption as compared to Cis-isomers.
4. Qualitative analysis: (Identification of unknown compounds) the detection of unknown
compounds is carried out by making the comparison of unknown spectrum with the known
spectrum.
5. By using this technique the structures of some vitamins and organic compounds can be identified.
6. By the use of this technique the no. of chromophores such as aldehyde, isolated double bonds
can be identified.
7. By this technique we can also perform conformational analysis and we can also determine the
rate of reaction.
8. By this technique we can also determine the quantitative analysis of drug having even low
concentration in the sample. The example of the drugs are paracetamol, vitamin B2 (riboflavin),
Vitamin C (ascorbic acid, Vitamin B12 cyanocobalamine.
9. We can also determine the concentration of the known compound if it fallows Beer’s Lambart’s
law.
10. We can also measure the kinetic measurement;
i. The rate of chemical reaction can be determined as, decrease in absorbance when
reactants are absorbing species.
ii. Increase in absorbance when products are absorbing species.
11. We can also determine the ionization constant of an acid and base by measuring the absorbance
at different concentrations.

Examples in aromatic systems:
In the poly nuclear hydrocarbons for example such as anthracine, naphthalene and benzene, anthracine
has highest λmax value because it has a highly extended conjugated system as compared to naphthalene
and benzene.
Benzene Naphthalene Anthracine
λmax (wavelength) 255nm 314nm 380nm
Ɛ (absorbance) 230nm 289nm 9000nm
Some of the pharmaceutical examples are as fallows;
Ibuprofen (tablet and suspension), Ponston (tablet and suspension), Vitamin syrup and dexamethasone
Injection.

Gas
Chromatography
&
Mass
Spectrometry

Chromatography
Mechial zwitt was the
chromatography is made
measurements or writing.
first scientist who discovered chromatography technique. The word up of
two Greek words ‘Khromatos’ means Color and ‘Graphy’ means
Definition:
It is a separation technique. In which the sample mixture is separated into its components by the use of
proper solvent.
Chromatography is a physical or chemical process or both;

It is a physical process as there is no chemical reaction or formation of new compound.


It is a chemical process as there is change in composition before and after the chromatography.

With chromatography we can get access to the composition of the mixture to be separated.
Composition consists of;
 Number of components
 Quantity of components
Chromatography is an analytical method used primarily for the separation of a sample of mixture. It
involves the distribution of the sample mixture between two phases i.e. stationary phase and mobile
phase.

Stationary phase:

It is may be a solid or liquid supported as a thin film on the surface of an inert solid. The phase
(solid / liquid) on which the mobile phase flow is called stationary phase.

Mobile phase:

The mobile phase flowing over the surface of the stationary phase that may be a gas or a liquid.
The solvent (gas / liquid) which is used to separate the components of the mixture is called
mobile phase.
Base of chromatography:
Polarity is the base of chromatography. We are trying to make unionized and polar drugs.
Order of polarity:
The order of polarity is the sequence of solvents with the increasing or decreasing order polarity.
Example: The given sequence of solvents form non-polar to polar.
n-Hexane > Petroleum ether > Chloroform > Ethyl acetate > Alcohol (Methanol > Ethanol > Propanol >
Butanol)
In case of alkanes increase in carbon no. decrease in polarity of alkanes. The most polar solvent is Water.

Gas Chromatography GC
Introduction:
It is an analytical technique. In this technique, liquid or gas
sample is used for the analysis. The particle size of the sample
mixture must be ranging 0.4µ – 0.5µ for the instrumentation.
In case of liquid sample it will be converted into vapors first
with the help of oven. The technique is used only when the
substance is heat stable because temperature is about 300o
C.
In this technique two phases are used;
 Mobile phase
 Stationary phase
Mobile phase:
The gas is the mobile phase. Normally Nitrogen N2 or Helium He gas is used as mobile phase. Only one
gas can be used at a time, both can’t be used simultaneously.

Nitrogen N2 is non-reactive in gaseous phase.


Helium He is an inert gas. Helium is mostly used because it shows good results.

Stationary phase:
Column in the form of coil is used as stationary phase. It is commonly made up or either metal or glass
tubing. There are two commercially available columns;
There are two types of gas chromatography on the bases of stationary phase;

Gas solid chromatography GSC


Gas liquid chromatography GLC

The column tubing contain solid silica. This column is used in Gas solid chromatography.
The column consist of two tubes. The outer tubing contain solid silica. The inner tubing contain either
polar polyethylene glycol or non-polar polysiloxane in liquid form depending upon the nature of sample
mixture. The column is used in Gas liquid chromatography.

Principle:
Polarity is the principle of gas chromatography. We will find the polarity of the stationary phase, and
the sample to be examined and separated.
Relation of Gas Chromatography with Fractional distillation:
Similarities; these both techniques depends upon the boiling points of the component mixtures.
Difference; Fractional distillation is used on large scale or macro scale. While, GC is used for Lab
scale purposes.
TLC:
TLC stands for thin layer chromatography.
Advantage: It is a cheap process.
Disadvantage: It has no temperature regulation.
HPLC:
It stands for High Performance liquid chromatography or High Presure Liquid Chromatography.
Advantages:

The results are precise.


Thermolaible substances can be analyzed by the technique.

Disadvantages:
Thermostable compounds are not analyzed up to 40o
C temperature.

GC working:
A volume of mixture which is to be separated is injected into the head of column by the syringe or micro
syringe. A mobile phase (gas) sweeps or carries the mixture into the column, this motion is inhibited by
the adsorption of each component of the mixture in the column. The rate at which the mixture or the
components of mixture progress or more along the column depends upon the strength of adsorption,
and the strength of adsorption depends upon the polarity of the mixture and the polarity of the
stationary phase.
So, in this way each component in the mixture becomes separated in the column as the mixture reaches
the end of the column at the different times (retention time).
Retention time: the time in between injection of the sample and detection of components.
Detector detect the components qualitatively and quantitatively. It calculates the retention time of the
different components.
Flushing or washing of the instrument is done only by gas. The flow of the gas is maintained to get good
results so that the sample to be separated moves smoothly through the column.
Selection of column:
It depends upon;
 Polarity of the mixture and polarity of the column.
 We also consider the thickness, diameter and the length of the column.

Column Temperature & Temperature program;
Column is placed in an oven the temperature of which is specially controlled. If there is no difference in
initial and final temperature of the whole process so this is called Isothermal process.
If there is difference in initial and final temperature so, the initial temperature, rate of increase in
temperature and final temperature is called as Temperature program.
Detectors:
Most commonly used detectors in GC is two;

Flame ionization detectors FID


Thermal conductivity detector TCD

Among the others are;

Catalytic combustion detector


Infrared detector



Atomic emission detector



Electron capture detector



Helium ionization detector



Photo ionization detector

Data analysis:
Two types of analysis are carried out by the GC;

Qualitative analysis: it refers to that which components are present in the sample mixture. The no.
of peaks gives the no. of components of mixture. For example; active ingredients.

If the retention time of the mixture is similar to the reference then quality test is performed.

Quantitative analysis: it refers to that how much active ingredient is present in the sample mixture.


The greater area of the peak indicate the quantitative analysis of the component.

Applications:
We can isolate the compounds which are thermostable by using GC, but GC alone is not sufficient or
efficient to separate the mixture. Therefore, GC is connected with Mass spectrometer MS to get more
accurate results.
We can connect MS with GC, LC, HPLC, TLC, and we can connect MS with MS.
Mass Spec can be combined with gas chromatography to analyze mixtures of compounds.
 GC separates the components of the mixture.
 Each component is analyzed by the Mass Spectrometer.

Introduction
Spectroscopy
It is the combination of two words spectrum & scope. Spectrum means, “a set of specific no. or set of
specific values or a set of digits,” while the word Scope means “Look or Watch.”
So, spectroscopy means, study of specific values or numbers. It deals with the study of electromagnetic
radiations (radiations possessing both the properties of electrical and magnetic nature). Electromagnetic
magnetic radiations include ultra violet, infrared, microwave, radio waves, cosmic waves, visible waves
etc.
Every light is a combination of different colors. If the spectrum obtained is from visible region then, it is
‘Photometry.’
Spectrometry:
It is the combination of two words spectrum & meter. Spectrum means, “a set of specific no. or set of
values or a set of digits,” while the word Meter means “measurement.”
So, Spectrometry means measurement of spectrum. When there is no absorption or emission of
electromagnetic radiations, then this study is known as spectrometry, but here the spectrum is not
made up by the electromagnetic radiations. The spectrum is Mass spectrum. If it has the concept of
mass then it is referred to as Mass spectrometer.
Mass Spectrometry
There is breakdown of a molecule into its different fragments that is why it is called as Mass spectrometry.
If a large molecule is present then, there will be formation of too many fragments. So, that’s why MS is
used in combination with other techniques.
Attachment of MS with other techniques
This is the only technique which is used in combination. Only MS is not so much efficient for separation
& identification of any molecule that’s why MS is attached to GC, HPLC, LC and TLC.
GC, LC, and HPLC firstly isolate compounds and then we connect them with MS and it is then attached
with digital library and compounds are identified.
It separates the components then the only one peak is obtained (make fragments of the compounds).
We can’t tell the mechanism of any drug without knowing its 3D structure. We want only one compound
to come out that is why MS is combined with others.

Example:
For example; In case of morphine the possible fragments are,
benzene, cyclohexene etc.
They all got separated and different fragments are formed. The
fragment which has maximum quantity in the parent compound
forms the first peak and it is called base peak (100% peak).
The mass is inversely proportional to the distance. Therefore, the whole compound is broken down and
then the quantity of various fragments is checked.
Basic Theory
Mass Spectrometry is the most sensitive technique for structural determination (elucidation). In MS the
molecule is broken down into fragments. These fragments are separated according to their mass to
charge ratio (m/e). Each fragment give its peak (response) on reaching to detector. These fragments are
recorded as Mass spectrum.
Mass spectrum is the graphical representation and comparison in between mass to charge ratio vs.
relative intensity. The highest peak is recorded as 100% which is also called as base peak. The other
peaks are recorded with respect to the base peak (100% peak).
The total energy is 70 eV. Some amount of energy (10 eV) is utilized to ionize the compound and the
remaining energy (70 eV) is utilized to break the bonds. MS break all bonds of different compounds and
fragments are formed (those compounds which have too much similarities).
If we give too much compounds to MS then it will mix all of them and we can’t identify the original
parent compound.
Highest peak (base peak) represents the highest quality.
Every fragment shows its specific response.
Detector provides a peak of every fragment. Detector detects the fragments on mass to charge ratio
(m/e) and their relative intensity use. Retention time is not checked over here as we use mass.
Recorder made comparison and it shows results. It is then attached with digital library. After
fragmentation the fragments are again combined to check the structure of the sample.

Instrumentation and Data analysis
There are five major parts of Mass
spectrometer, which are as fallows;
1. Ion source
2. Mass analyzer
3. Vacuum Pump
4. Detector
5. Recorder
Schematic diagram of MS:

1. Ion source:
It is also called ionization chamber or ionic chamber or ionization source. Ionization of the sample occurs
in ionization chamber.
Ionization potential: The minimum energy required to ionize an atom or molecules is called Ionization
potential. The energy may be supplied by different ways depending upon the physical or chemical
nature of sample.
Methods of ionization
Several methods are employed for the ionization of the sample depending upon the physical state and
chemical nature of the sample.
The ionization methods which are used are discussed below;
i. Electron impact EI
The first and the common method of ionization of Electron impact or electron ionization. In this method
electron beam is used for the purpose.
The sample is volatilized in a separate chamber and vapors at pressure 10-4
– 10-6
torr are allowed to
enter in the ionization chamber.
In the ionization chamber there is a bombardment of electron beam which can remove or eliminate
electron from sample creating Radical cations. The source of electronic beam is electrically heated
filament of tungsten.
On the opposite side of filament there is an anode plate which acts as “Electron trap.” Electron are drawn
towards trap and travel across the chamber with the help of magnates called as “Collimating magnates.”
The time span for the production of ions 1 – 5 µs. Ions which are produced move towards mass analyzer.
Detector signals are directly proportional to the no. of ions hitting to the detector. By adjusting the
magnates, ions of all the masses are collected and countered.

ii. Chemical ionization CI
In chemical ionization a reagent (Methane, Ethane, Butane or isobutene) is introduced into high
pressure source (0.1 – 1.0 torr) and ionized by electrons bombardment.
Primary ions are produced.
CH4 + e- CH4
+. + 2e
Due to the high pressure there is a possibility or colliding a primary ion with the other molecules and the
reaction will be like;
CH4
+. + CH4 CH3
. + CH5
+
The introduction of small amount of sample in the vapor phase in the ion source results in the reaction
between reagent ions and sample molecules.
There might be hydrogen ion H+
transfer reaction.
CH5
+ + MH MH2
+ + CH4 (M + I)+
There might be hydride ion H-
transfer reaction.
C2H5
+ + MH M+ + C2H6 (M - I)+
These reactions are exothermic I n nature. The energy of exothermicity acts as internal energy which
may lead to fragmentation of the sample molecule.
iii. Field ionization FI
It is also known as Field desorption FD. Many organic compounds are thermally unstable to meet the
requirement of electron ionization or chemical ionization, they can be ionized by Field ionization.
Sample in the vapor phase can be ionized when the molecules of the sample pass near the metal anode
carrying electric field of 1010
V/m. The electrons e- are sucked or eliminated from the sample molecules
into the metal and the resulting molecular ions are repelled towards the cathode slit. These molecular
ions are passed towards mass analyzer.
In FD or FI, sample in the vapor phase is directly deposited on metal anode and high electric field
produces ionization, fragmentation and desorption.
Application:
This method is used for the molecules having high molecular weight or naturally occurring complex
compounds. For example Carbohydrates.

iv. Fast Atom Bombardment FAB
In this method the sample is dissolved in a viscous liquid typically Glycerol (matrix material) and
ionization is achieved by the bombardment of sample matrix by the beam of fast moving neutral atoms.
The bombarding atoms are mostly inert gases or Nobel gases, like Xenon (Xe) or Argon (Ar).
In order to achieve a very high kinetic energy K.E, the atoms are fast ionized and then these ions are
pass through electric field.
After getting accelerated these ions enter into a chamber containing neutral atoms of the same gas and
there will be collision of ions and neutral atoms which result in the exchange of charge.
Xe+. (Fast) + Xe (Thermal) Xe (Fast) + Xe+. (Thermal)
2. Mass analyzer:
It is also called as ion separator. Following are the commonly used mass analyzers in Mass spectrometer;
i. Magnetic sector analyzer
ii. Time of flight
iii. Quadrupole mass Analyzer
i. Magnetic sector analyzer:
It is further divided into two types;
 Single focusing analyzer
 Double focusing analyzer


 Single focusing analyzer:
It is a curved metallic tube placed between two poles of electromagnet or magnet with a specific
magnetic field.
When an ion contains a charge Z is repelled by electrostatic field. Its potential energy is ZV due to
repulsion. Its potential energy is converted into Kinetic energy i.e. 1/2mv2
.
When ions enters into magnetic field, magnetic field exerts centrifugal force on ions which is equal to
mv2
/r. Magnetic field also exerts centripetal force which is equal to ZHv. At equilibrium the two forces
becomes equal.
The instrument in which ions are formed analyze and separated only by the application of magnetic field
is called single focusing Mass spectrometer, and the analyzer is called as single focusing analyzer.

By comparing the K.E and P.E;
. = .
12 2 =
2 = 2
By comparing the centripetal and centrifugal force;
2 =
=
2 = 2 2 2
2
By comparing equation (i) and equation (ii);
2 2 2
= 2
2
2 2
= 2
2 = 2 2
By inverting the equation;
2 2 = 2
∝ 2
It is clear from the above expression that mass to charge ratio (m/z) depends upon the radius of curvature.
 Double focusing analyzers:
It is an instrument in which electrostatic analyzer is used in addition to magnetic analyzer. Electrostatic
analyzer focuses or directs the ions towards the magnetic analyzer. For better results or resolution
monoenergatic (with same Kinetic energy) can be selected or can be obtained by magnetic analyzer.

ii. Time of flight:
The method of analysis deals with the measurement of time required for an ion to travel from the ion
source to detector.
 The sample is volatilized into the space between the first and second electrode and a burst of
electrons is allowed to produce ions. An extraction potential (E) is applied for another short time
period which will result in the ions being focused.

 After focusing, accelerating potential is applied (V) for a much shorter period than that used for
ion production. So that all the ions in the ion source are accelerated simultaneously.

 The ion is then pass through the third electrode into the drift zone and are eventually collected
by the sensing electrode.
1
= (2 )2
 Since all the ions have started from the ion source and gained the same K.E (1/2 mv2
= zeV). During
acceleration they began to separate according to their mass as they drift along the flight tube and
ions of different masses arrive at the detector at different times as per above equation.
iii. Quadrupole Mass analyzer:
Quadrupole mass spectrometer is compact rugged and easy to operate and consequently is a popular
instrument in use.
 Quadrupole mass spectrometer consist of four rods which must be straight and parallel and so
arranged that the beam of ions is directed axially between them.

 A voltage comprising a DC and radiofrequency component is applied between adjacent rods,
opposite rods being electrically connected.

 Ions are accelerated into the center between the rods with a relatively small potential ranging
from 10 to 20 volts.

 Quadrupole mass analyzer is a mass filter. Combined DC and RF potentials on the quadrupole
rods can be set to pass only a selected m/e ratio.

 All other ions do not have a stable trajectory through the quadrupole mass analyzer and will
collide with the quadrupole rods, never reaching to the detector.

3. Detector:
Three types of commonly used detectors are discussed below;
i. Faraday cup detector
ii. Photographic plate detector
iii. Electron multiplier detector
i. Faraday cup detector:
It is a metallic cup which is maintained at potential which allows the ions to be captured by the cup.
ii. Photographic plate detector:
It is cupped at right angle to path of ions. So that, there is a linear formation of images and abundance
of ions is determined by the intensity of each image.
iii. Electron multiplier detector:
It is a series of electrodes (dynodes) which are connected to each other. When ions hit the first dynode,
there is a release of large no. of electrons. These electrons hit the second dynode and then it will release
large no. of electrons. This process goes on throughout the series of electrodes, which are normally 10.
As a result of this sequence there is a production of electric current which is amplified and presented in
the form of graph. The graph is called as Mass spectrum.
4. Vacuum system:
All mass spectrometers operate at low pressure, just to avoid the ion collision. Any collision of ions may
resulting ionic reactions; neutralization and scattering.
To minimize the collision whole procedure is operated at low pressure or high vacuum (10-4
– 10-8
torr).
The vacuum system also attracts the ions towards the detector.
5. Recording of Mass spectrum:
The most important method of recording the
mass spectrum is the use of online computer.
The whole system is known as data system.
A printer is also attached to the data system to
take out the print of recorded mass spectrum.

Applications of Mass spectrometer
MS is one on the most sensitive technique for the qualitative analysis therefore it has various uses. The
various applications of the Mass spectroscopy are discussed below;
1. Preparation of isotopes:
MS is very useful for preparations of pure isotopes, high polymers and natural products can be analyzed
by this.
2. Cis-trans isomers:
It is also used to distinguish between Cis & Trans isomers, since stability of ions produced may differ for
Cis & Trans ions significantly.
3. Study of free radicals:
It is useful to study free radicals, determination of bond strength, evaluation of heat of sublimation etc.
4. Study of closely related compounds:
It is useful in the analysis of closely related compounds. For example; hydrocarbons, petroleum,
products, lubricating oils etc.
5. Identification of Molecular formula:
It provides important information for identification by help of molecular weight, molecular formula and
by fermentation pattern.
6. Determination of molecular weight:
It is best tool for the determination of molecular weight of the substance (where substance is
bombarded with moving electrons and its mass spectra is recorded, the mass of peak at highest m/e
reveals molecular mass accurately also the molecular formula.
7. Trace analysis:
The inorganic trace analysis MS can used for trace analysis of elements in alloys and minerals and in
super conductors.

Electrochemical
Methods
Potentiometery, Polarography and
Radiochemical techniques.

Potentiometery:
It is one of the volumetric technique of electro analytical chemistry which is used to measure electrochemical
potential of charged particles. An electrode system which is connected to potentiometer used to measure
this potential can detect ions while, other substances also present in the solution.
Measurements are always made when no current or very little current passes through the
potentiometric circuit. So, the composition of the solution measured is not changed making quantitative
analysis possible. So, potentiometric method involves two types of electrochemical analysis;

1st
method is used for the determination of pH based upon effect that difference of potential
between two suitable electrodes dipping into a solution containing hydronium ions which
depends upon concentration or activity of hydronium ions.



2nd
category is potentiometric titrations which involves the measurements of changes in EMF of
the cell by adding a titrant (that is monitoring of the potential serves only to locate equivalence
point for a titration).

So the measurements with electrochemical cells used to calculate ions concentration in solutions by
electrode potential using Nernst equation which was discovered in the late 1800’s.
Types of Electrodes:
Electrodes used for measurements in potentiometery includes;
1. Reference electrode
2. Indicator electrode
Electrode used for reference electrode are usually includes Hydrogen ion electrode, Saturated calomel
electrode SCE, Mercury sulfate and Silver Chloride electrode. While, the indicator electrode is exposed
to the analyte solution and its potential varies depending upon the concentration of ions present in the
solution.
Each electrode is placed in a separate solution and connected to a single potentiometer, while, a salt
bridge is exposed to each sample completing an electrical circuit.

Electrode potential E (volts):
A cell is made up of two parts or two half cells, each containing an electrode in each half cell. There exist
a difference of potential between electrodes and the solution in which it is dipping and this potential is
known as Electrode Potential E.
The electrode potential depends upon;
 The nature of electrodes
 Concentration of solution
 Temperature
When temperature is 25o
C and the solution is one molar (which is unit activity for all species) or one
atmospheric partial pressure in case of gases than this potential is known as Standard Electrode
Potential Eo and it is measured in Volts.
Application:

It is used for the determination of thermodynamic cell potential. For example; EMF.


It is used for the calculation of equilibrium constant for redox reaction.

Limitations:
Electrode potential has certain limits, which are as fallows;
 Electrode potential will predict whether a given chemical reaction occur or not, but they
indicate nothing about the rate of reaction and also don’t assure the success of reaction.

 They are useful to predict that a reaction will not occur if the potential differences are not
sufficient.
Diagram:

Examples:
1. Half-cell reaction:
Zn(s) Zn+2
+ 2e-
Eo
= 0.76 volts (oxidation)
Half-cell reaction:
Cu+2
(aq.) + 2e-
Cu(s) Eo
= 0.34 volts (reduction)
Overall Cell reaction:
Cu+2
(aq.) + Zn(s) Cu(s) + Zn+2
Fe+2 e- + Fe+3
Eo
= 0.771 volts (oxidation)
Half-cell reaction:
Ce+4
+ e-
Ce+3 Eo
= 1.61 volts (reduction)
Fe+2
+ Ce+4
Fe+3
+ Ce+3
Ni(s) Ni+2
+ 2e-
(oxidation at anode)
Half-cell reaction:
Fe+3
+ 2e-
2Fe+2
(reduction at cathode)
Ni(s) + 2Fe+3
Ni+2
+ 2Fe+2

Measurement of pH
It is based on the fact that difference of potential between two suitable electrodes dipping in a solution
congaing H3O+
or H+
ions. It depends on the concentration of or activity of H3O+
or H+
ions.
The development of potential is not a specific property of H3O+
. The solution of any ion will develop a
potential directly proportional to the concentration of that ion, if a suitable pair of electrode is placed in
a solution.
The determination of potential of cell or half-cell under the conduction that no current flows through it
and potential across two electrodes is measured by potentiometric methods.
The electrode whose potential depends upon relative concentration of ion to be determined is called
indicator electrode.
pH:
Acidity or alkalinity of a reaction is the most important factor as it controlled the;
 Rate of reaction
 Nature of species present
 Even taste
pH can be defined as, “ the negative logarithm of hydrogen or H+
ion concentration.”
1
= − log[ +] = log [ +]
Similar notation can be applied to OH- ion concentration as;
1
= − log[ −] = log [ −]
On thermodynamic ground, these reactions are modified as;
= − log +
= − log −
a H+
= is the activity or the effective concentration of H+
ions.
a OH- = is the activity or the effective concentration of OH- ions.
For dilute solution;
a H+
= [H+
]
a OH
-
= [OH-
]
So, the pH is the measurement of the activity of the H+
or H3O+
ions.
Activity of the substance means, its effective molar concentration.

Molar concentration:
It refers to mol/ liter of solution.
= − log[ (a +)]
= − log[ (a 3 +)]
pH scale:
Role of Buffer:
As many reactions depends upon the concentration of H3O+
ion. It is important to control the pH. This is
usually achieved by solution which have definite pH to retain pH for long time and its pH is not affected
by addition of bases or acids or even with dilute is known as buffer solution.
It should be noted that weak bases and their salts acts as buffer. Some standard buffers are;
Standard Buffers pH at 25o
C
0.1 M NH3 / 0.1M NH4Cl 9.25
0.025 M NaHCO3 10.012
0.025 M Na2CO3 10.012
0.1 M Borax 9.180

NERNST Equation:
The equation was given by Walther H. Nernst, he was awarded Nobel Prize in 1920.
The NERNST equation enables us to determine the;
 Electromotive force EMF of many processes
 Cell potential of a system called Galvanic cell
 Energy of a chemical reaction
The energy of a chemical system drives the charge to move and driving force give rise to cell potential
therefore all these relationships are tied together in the concept of NERNST equation.
(Reactants) A + B C+ D (Products)
= − ln
[ ] [ ]
[ ] [ ]
As we know; ln= 2.303 log10
= −
2.303
log10
[ ] [ ]
[ ] [ ]
By putting the values of R, T and F;
2.303
=
0.591
And,
[ ] [ ]
[ ] [ ] = = ReactionQuotient
So,
= −
0.591
log10
As, log10 = 1 so, final equation will be;
= −
0.591
Where,
 E = Electrode potential
 Eo
= Standard Electrode potential
 R = General gas constant 8.313 JK-1
mol-1

 T = Temperature
 n = No. of moles electrons appears in half cell
 F = Faraday’s Constant 9.648 X 104
JV-1
mol-1

 In = Natural logarithm 2.303 log10

NERNST equation for redox reaction:
A reaction in which both the oxidation and reduction is taking place is called oxidation-reduction
reaction or Redox reaction.
The NERNST equation for the redox reaction is as following;
General reaction
A ox + ne-
B red
As NERNST equation is;
= −
0.591
log10
So the equation for redox reaction will be,
= −
0.591
log10
Example:
In the following reaction;
Ag+ + e-
Ag
The NERNST equation will be;
= −
0.591 log10
[ ]
[ +]

Methods of calculation of pH
1. Calorimeter method
2. Indicator method
3. pH meter method
4. Glass electrode pH method
pH Meter
It is a device which is used for the determination of pH of solution.
Principle:
The pH of the solution may be determined by measuring the potential difference between a pair
of electrodes immersed in the solution.
One of the electrode is an indicator electrode (e.g. glass electrode) and other one is a reference
electrode (e.g. calomel electrode).

Reference Electrodes
A reference electrode is a half-cell having a known electrode potential that remain constant and is
independent of the composition of the analyte solution. Reference electrode is always treated as anode.
Properties of an ideal Reference electrode are as fallows;

It must have a potential that is accurately known and constant.


It should be completely intensive to the composition of analyte solution.



The electrode must be rugged or easy to assemble.



It should maintain a constant potential while passing a small amount of current.

Commonly used reference electrodes are mentioned below;
1. Silver-silver electrode
2. Saturated calomel electrode (SCE)
3. Mercury (I) sulfate electrode
4. Standard Hydrogen electrode (SHE)
1. Silver-Silver Chloride Electrode:
A reference electrode system analogues of the calomel electrode consist of AgCl2 immiscible in a
solution of KCl that is also saturated with silver nitrate (AgNO3).
Construction:
The electrode is contained in a Pyrex tube fitterd with 10mm fritted
glass disc or porous drug. A plug of agar gel saturated with KCl is
formed on top of the disc to prevent loss of solution from half-cell.
The plug can be prepared by heating 4g – 6g of pure agar in 100ml of
water until solution is completely form and then add about 35g of KCl.
A portion of this suspension is poured into tube upon cooling it solidify
to a gel with low electrical resistance.
A layer of solid KCl is placed in the gel and tube is filled with saturated
solution of salt. 1 to 2 drops of 1M silver nitrate is then added and a
heavy gauge 1mm – 2mm diameter. Silver wire is inserted in the
solution. The potential of electrode is governed by activation of
Chloride ion in KCl solution.
Reaction:
AgCl (s) + e- Ag (s) + Cl-(aq)

2. Saturated calomel Electrode:
It is mostly used by analytical chemists as it can be prepared easily.
Construction:
It consist of a 5cm – 15cm long tube that is 0.5 – 1cm in diameter.
An Hg/Hg (I) chloride paste is saturated in Potassium chloride KCl
is combined in an outer tube and through a small opening. An
inert metal electrode is immersed in the paste contact with
analyte solution, is made through a fritted disc. A porous fiber or
piece of porous sealed in the end of other tubing.
The conc. Of calomel electrode depends upon the conc. Of KCl in
cell, the usual conc. Of KCl used as 0.1M, 1M and saturated
solution but saturated solution is preferred. It is known as
saturated calomel electrode.
Chemical reactions in calomel half-cell:
It has larger temperature coefficient than the other Reference electrodes. It is only important in those
circumstances when temperature changes occur during measurements.
Hg2Cl2 + 2e-
2Hg (l) + 2Cl-1
(aq)
2Hg + 2Cl-1
Hg2Cl2 + 2e-
The potentials of SCE vs. normal Hydrogen electrode in saturated molar.
Application:

It is used to determine the unknown pH.


It is most widely used because of the consistency of its potential and ease of preparation.

3. Mercury (I) sulphate electrode:
Principle:
A requirement of a reference electrode is that the potential be fixed and stable unaffected by passage of
small amounts of current required in making potentiometric measurement.
Construction:
It is similar to saturated calomel electrode but difference is that it utilizes H2SO4 (0.05M) saturated
Mercury (I) sulphate.
Application:

It is used in solution where lead (Pb) or silver (Ag) ions are present and has potential of 683 mV.


Indicator Electrodes
An indicator electrode is an electrode system having a potential that varies with variation in the
concentration of analyte.
Characteristics of an ideal Indicator electrode are as fallows;

An ideal indicator electrode respond rapidly.


Reproducibly, it can bring changes in the concentration of an analyte group or ion.

Limitations:
Although, no indicator electrode is absolutely specific in its response a few are now available that are
remarkably selective.
There are three types of indicator electrodes;
1. Hydrogen electrode
2. Glass electrode
3. Ion selective electrodes
Standard Hydrogen electrode SHE:
It was extensively used in early electrochemical studies;
Construction:
It consist of a platinum electrode immersed in an electrolyte solution. The electrolyte solution is HCl of
unit activity that is kept saturated with Hydrogen gas. A saturated condition is maintained by bubbling of
hydrogen gas continuously through solution that immediately absorb to the electrode surface.
Platinum black: a piece of platinum foil is coated with Platinum black. It provides large surface area for
adsorption of hydrogen gas.
Half reaction:
The half-cell reaction can be written as;
H+
+ e-
½ H2
Or
2H+
+ 2e-
H2 (aq)
H2 (aq) H2 (g)

Glass electrode:
For pH measurements only a bulb is needed to be submerged (dipped in the analyte solution whose pH
is to be determined)
There is internal reference electrode and electrolyte (Ag/ AgCl/ Cl-
) for making electrical contact within
the glass membrane and its potential is constant set by the concentration of HCl.
A complete cell is then represented as fallows;
Reference electrode H+
Glass H+
Reference electrode
(external) SCE Unknown Membrane Internal (internal)
The double line represents the salt bridge of the reference electrode.
The glass electrode is attached to the indicating electrode terminal of the pH meter. While, the SCE is
attached to the reference terminal. Then the potential of the glass membrane is given by the equation;
= −
2.303
log10
+ ( )
… … … … … … … ( )
+ ( )
And voltage of the cell is given by;
= + 2.303 log10
+ ( ) … … … … … … … ( )
Where;
K = is the constant that indicates the potential of the 2nd
reference electrode.
a = activity
1. Liquid Junction potential
2. A potential at the glass membrane due to H+
(internal) and this potential is known as,
“Asymmetric potential.”
Working:
Glass electrode induced an ion exchange reaction. So, at both inner
and outer surfaces of the glass membrane absorb water and form
hydrated layers in the glass structure. The hydrogen ions diffuse
through the hydrated layers, in the direction of lesser
concentration and in the process replace Na+
ions and other
metallic ions contained in this structure of glass membrane.
Net result of this diffusion and ion exchange process a plot
boundary potential is set up on each side of the glass membrane.

Precaution:
The glass membrane electrode will not work properly it its membrane is not hydrated. So it should be
stored in distilled water to avoid dehydration.
Advantages:

Operative over a wide pH range.


It is not affected by oxidizing or reducing agents.



Fast responding and function well in physiological systems.

Disadvantages:

It is extremely fragile


Most of the glass electrodes have a high resistant. So that they cannot be used with simple
potentiometers.

Applications:

Due to its convenience it is almost universally used for pH measurements.


Commonly used for the determination of H+
ions concentration.

Ion selective electrode ISE:
They consist of a sensor electrode that convert the activity of
specific ion dissolved in a solution into an electrical potential,
which can be measured by pH meter or voltmeter.
The sensating part i.e. the electrode is usually made by an ion
specific membrane along with reference electrode.
Advantages:

Responsive over wide concentration range i.e. 0.1 – 10,000ppm.


Electrode bodies are shock proof.


Electrode bodies are chemically resistant.



Sample analysis can be complete within 1 –2 minutes.

Types:
There are three types of ions selective membranes which are used in ion selective electrode;
1. Glass membrane (It has selectivity for single charge ions over H+
, Na+
, and Ag+
.)
2. Crystalline membrane (It has selectivity for both cations and anions.)
3. Ion exchange resin membrane (It is the most popular one. Its selectivity changes by changing the
composition of resin).


Ion exchange resin:

It has five types;
1. Solid state electrode
2. Coated wire electrode
3. Liquid-liquid electrode
4. Plastic membrane electrode
5. Potentiometric electrode (enzymes based electrode to measure the substrate)
Solid state/ crystal membrane electrode:
The most typical and successful example is fluoride electrode. The selective electrode is one of the most
successful and useful electrode since determination of fluoride ion is difficult by other electrodes.
Construction:
Membrane consist of single crystal of Lanthanum
Fluoride doped with Chromium II to increase the
conductivity of the ion. This electrode has at least
1000 folds selectivity for fluoride ion over
chloride, bromide, iodide, nitrate, sulfate, mono
hydrogen, bicarbonate and phosphate ions.
A useful solution of minimizing hazards with
fluoride electrode consist of a mixture of an
acetate buffer having pH 5 – 5.5, this solution of
NaCl and cycloxylene dinitro acetic acid (CDAA)
available in market as Tisah.
Coated wire electrode:
These electrodes are very convenience to prepare and use. It can be prepared by coating a wire with
PVC membrane.
Coated wire ion selective electrode have no internal reference solution and consist of PVC matrix sensor
system coated onto a metal wire (Pt, Ag, and Cu).
For example; Nitrate coated ISE.
Advantages:

It responses near to NERNSTian.


It is easily made.


Liquid-liquid electrode:
Construction:
Potential determining membrane is a layer of water
immiscible liquid ion exchanged held in place by an
inert/ porous membrane. Porous membrane allow
contact between the test solution and ion exchanger
but minimize mixing. It is either a synthetic, flexible
membrane or a porous glass frit.
Internal filling solution contains;
 The ions for which ion exchanger in specific
 A halide ion for the internal reference
So, the filling solution contains usually a chloride salt of a primary ion. The chloride established the
potential of the internal Ag/ AgCl electrode. CaCl2 for Ca+2
electrode and KCl for K+
electrode.
Example:
The common example is Ca+2
selective electrode, ion exchanger is Ca+2
organophosphorous compounds.
Selectivity:

Selectivity of this electrode is 3000 for Ca+2
over Na+
or F
-
, similarly 200 for Mg+2
and 70 over Sr+2
.


It can be useful or used over the pH range of 5.5 – 11.0



A divalent ion exchange electrode that respond to several cations is also available.



It responses equally for Ca+2
& Mg+2
and useful for measuring water hardness.


A copper and lead electrode are also available. Anion selective electrode of this type are
available for nitrate, perchlorate and chloride, same in principles except that a liquid anion
exchanger is used instead of cation exchanger.

Plastic membrane Ionophore electrodes:
A very versatile and easy to prepare in which a neutral lipophilic (organic loving) ionophore that selectively
complexes with the ion of interest is dissolved in a soft plastic membrane. The ionophore should be lipophilic
so that it is not leached (broken/ leak) from the membrane upon exposure to aqueous solution.
Construction:
Plastic membrane usually Poly vinyl chloride (PVC) based and consist of about 33% PVC and 65%
plasticizer.
Example:

K+
ion selective electrode incorporation the ionophore valinomycin (naturally occurring antibiotic),
its selectivity for K+
ion is about K+
for that Na+
.


Potentiometric Electrodes (enzyme based electrodes to measure the
substrates)
Example:
Urea electrode is one of the good example.
Construction:
Urea electrode can be prepared by immobilizing urease in a gel and coating it on the surface of a cation
sensitive type glass electrode (respond to monovalent cations).
Working:
When electrode is dipped in a solution containing urea, the urea diffuse into the gel layer and the
enzyme catalyzes its hydrolysis to form ammonium ions.
The ammonium ions diffuses to the surface of the electrode where they are sensed by the cation
sensitive glass to give potential reading.

Potentiometric Titrations
It involves the measurement of an indicating electrode potential against a reference electrode and
plotting the changes of this potential difference against volume of titrant used.
It can also be understand as; Potentiometric titrations involves the measurements of potential of a
suitable indicator electrode as a function of titrant volume.
Example:
Direct measurement of 0.1M solution of HCl and acetic acid yield different hydrogen ion concentration
because the latter is particularly dissociates.
Comparison:

Compared with titrations that use chemical indicators the potentiometric end points provides more
reliable data.


It is particularly useful for the titration of colored and turbid solution.


It is useful for detecting the presence of unsuspected species in a solution.



The main disadvantage is that it is more time consuming than those titrations using indicators on the
other hand they are readily automated.

Process:
 The process usually involves measuring and recording the cell potential (in units of millivolts or
pH) after each addition of the reagent.

 The titrant is added in large increments at the outset. These volumes are made smaller as the
end point is approach sufficient time must be allowed for the attainment of equilibrium after
each addition of reagent.
Apparatus for Potentiometric Titrations

End point determination:
Several methods can be used for the determination of end point. The most straight forward method
involves a direct plot of potential with respect to reagent volume.
As fig 2 the midpoint is the steep rising position of the curve is the estimated visually and taken as the
end point.
Various graphical methods have been proposed to aid in the establishment of the midpoint but is
doubtful that these procedures significantly improves its determination (equivalence point).
For example;

First Derivative method


Second derivative method

Note:
Various titrations can be performed by this method;
 Acid base titrations.
 Redox titrations.
 Precipitation reaction titrations
 Complex formation titrations.
 Differential titrations.
Applications:

Assay of Caffine.


Assay of phenobarbitone.



Assay of thonzylamine hydrochloride.



Assay of tetrahydrozoline hydrochloride.


Polarography:
It is an electrochemical method of analysis based on the measurement of current how resulting from the
electrolysis of a solution at polarizable microelectrode as a function of applied voltage.
Electrodes used:
Two electrodes are placed in the solution;
1. One has fixed potential (voltage) or reference microelectrode is Saturated Calomel electrode SCE.
2. The other is indicator electrode which is Dropping Mercury electrode DME.
Polarography offers a new and exciting analytical tool that allowed for the first time for the
measurements of concentrations at submilimolar level without heroic efforts. So, the normal
concentration range from substances being analyzed is from 10-2
molar – 10-5
molar.
Process:
As the voltage applied to cell increases only a very small residual amount of current flows until the
substances under assay or under course, undergo reduction or oxidation. Then, the current increases at
first gradually then almost linearity with voltage and the resulting current or voltage current is called as
Polarograms.
Half wave potential:
It is oxidation or reduction potential at the polarographic curve that permit qualitative identification of
the reactants.
 It is a characteristic of electrolyzable species and is independent of concentration and related to
standard electrode potential Eo value.

 It is related to the form which is oxidizable or reducible molecule increase and can be shifted by
varying the pH of solution.
Fig: Showing half wave potential of polarographic circuit and polarographic curve.

Example:
Two compounds whose half wave potential overlap at a certain given pH can be resolve by varying the
pH or by adding a complex agent.
In acid solution the half wave potential of Tin (Sn) and Antimony (Sb) are -0.47 volts and 0.20 volts
respectively but, in alkaline solution they are -1.1 volts and 1.8 volts. The later condition preferred for
determination of these ions in the given mixture.
Electrodes:
In polarography the indicator or polarizable electrode which is mostly used is Dropping Mercury
electrode for reduction reaction. In few cases a Platinum electrode (stationary or rotatory) is used for
the oxidation reaction.
The curves produced by these reactions are known as cathodic and anodic waves respectively.
Applications:

The technique is useful for biochemical analysis.


The method is used for the analysis of inorganic species.



Most of the cations are reduced at DME includes the ions of Alkali or Alkaline earth metals.



The methods is also applicable for the determination of such ions as; bromate, iodate, vandate,
dichromate, nitrite etc.

Precautions:
The dissolved oxygen in any solution to be analyzed polarographically often interferes with accurate
determination of other species. The removal of oxygen before analysis is necessary using vacuum or
purging with Nitrogen N2 for several minutes.
Oxygen interference is due to production of two equal oxygen waves. The first is produced by its reduction to
Hydrogen peroxide H2O2, and second by further reduction of Hydrogen peroxide H2O2 into water.

Dropping mercury electrode:
The dropping mercury electrode (DME) is a working electrode made up of mercury and it is used in
polarography.
Construction:
A flow of mercury passes through an insulating capillary producing a
droplet which grows from the end of the capillary. It is produced in a
reproducible way. The capillary tube is of about 10cm – 15cm in length.
The Internal diameter of the capillary is about 0.05 mm.
Each droplet grows until it reaches a diameter of about 0.5mm and
releases. A vertical distance being maintained between DME and the
solution. The released droplet is no longer in contact with the working
electrode whose contact is above the capillary. Drop time is maintained
between 1 to 5 seconds. As the electrode is used, mercury collects in the
bottom of the cell.
Advantages:
Mercury is the hero of polarography. It has several advantages which are mentioned below;

Surface is reproducible, smooth and continuously renewed.


Hg forms soluble amalgam with many metals hence lowers their reduction potentials.



High over voltage of hydrogen on mercury makes possible the deposition of ions difficult to reduce
in aqueous solution e.g. alkali metal ions.


The surface area can be calculated from the weight of the drops.


Diffusion current assumes steady value.

Limitations:
DME has its certain limitations;

Mercury is costly and poisons.


Current passing through the cell increases as drop grows and decreases as drops breaks.



DME electrode generates some current like Residual current, migration current, kinetic current which
add error in current measurement.


Mercury is oxidized, it restricts the use of electrode as anode.


Radiochemical Techniques:
Radiochemical method of analysis is of five different types;
A. Isotope dilution analysis IDA
i. Direct IDA
ii. Indirect IDA
B. Neutron activation analysis
C. Radiometric analysis
D. Autoradiography
E. Autoradiography
All these methods are discussed in detail below;
A. Isotopes dilution analysis IDA:
A known amount of compound tagged or labelled with radioactive compound is added to the unknown
labelled compound and both are thoroughly mixed.
Pure unlabeled compound (weight) can be calculated by formula / equation;
= ( − 1)
Where;

W = Weight of the unlabeled compound to be determined


Wo = Weight of added labelled compound.



S = Specific activity of isolated pure compound



So = Specific activity of added labelled compound.

Inverse / reverse IDA:
“A known quantity of non-labelled compound is added to unknown amount of labelled compound
whose amount is to be determined.”
Addition of large quantity of unlabeled compound facilitates the isolation and purification of compound.
Application:

Method is useful for metabolic studies of labelled drugs.

Example: Benzoic acid content of camphorated tincture of opium BP.

B. Neutron activation analysis NAA:
The basis of this method is that, the element to be determined is bombarded with neutrons and
measure the activity of radioactive element.
75As + 1n0 nuclear reactions 76
As + γ
By the use of this method 75
As can be determined and measure radioisotopes 76
As so produced.
Sources of neutrons:

Nuclear reactor: It may produce neutrons flux up to 1014
Neutrons cm2
/sec.



Particle accelerator: The mixture of beryllium contained in a block of paraffin produces a flux of
neutrons up to 107
Neutrons cm2
/sec.

Uses:

The analysis is used for the determination of cadmium Cd in food products:


The analysis is used for the determination of arsenic As in sea food.



The analysis is used for the determination of mercury Hg in fish.



The analysis is used for the determination of Nitrogen N2, Oxygen O2, Fluorine F2, in organic compounds.



The analysis is used for the determination of metal ions in water (mercury, arsenic, zinc, cadmium,
antimony).

Advantages:

The method is very sensitive and it can measure quantity even in micrograms (µg).

Disadvantage:

This method is very specific but sometimes interferes from other elements as bromine Br and selenium
Se.

C. Radiometric assay:
In this analysis the radioactive substance is used indirectly to determine the quantity of an inactive
compound.
Example:
The determination of chloride ions by precipitation with radioactive silver 110
Ag.

D. Radio immune assay RIA:
This method is based upon the completion between radiolabelled antigen (Ag*) and unlabeled antigens
(Ag) for binding to a specific antibody (Ab) serum.
Ag* + Ag + Ab [Ag*Ab] + [Ag Ab] + [Ag* + Ag]

Ag* = Radiolabelled antigen


Ag = Antigen



Ab = Antibody



[Ag*Ab] = Radioactive antigen-antibody complex



[Ag Ab] = Non-reactive



[Ag* + Ag] = unreacted

The complex is separated and its radioactivity is measured.
The method can also be useful for quantitative analysis of various drugs, steroids, and hormones.
Drugs Steroids Hormones
Diazepam Estrogen Adrenocorticotrophic hormones ACTH
Nicotine Progesterone Follicle stimulating hormone FSH
Penicillin Corticosteroids Glucagon
Morphine Growth hormones
Gentamicin Insulin
E. Autoradiography:
The method is used for the detection of separated compounds on the surface of thin layer plate or
paper can be done by this technique.
Example:
16 different amino acids can be located and separated on thin layer plate by this method.

Differential
Scanning
Calorimetry

Differential scanning calorimeter DSC
Historical background:
The technique was developed by E.S. Watson & M.J.O.
Neill in 1962, but it was first introduced commercially in
1963 in Pittsburgh conference on Analytical chemistry &
Applied spectroscopy.
1st DSC that could be used in biochemistry was developed
by P.L. Peivalor and D.R. Moraselidze in 1964.
Definition:
DSC is a thermoanalytical technique in which difference
in amount of heat to require to raise the temperature of
a sample and reference is measured as a function of
temperature/ time. Both the sample and reference are
maintained at same temperature throughout the
experiment.
Introduction:
When heated to enough temperature all material undergo physical or chemical changes. These changes
alter the enthalpy or heat capacity of material, which in turn result in release or absorption of heat by
material. By detecting instantaneous rate of heat flow DSC provides quantitative thermodynamic and
kinetic information about physical & chemical changes occur in material.
DSC are able to measure amount of heat absorbed or released during such transitions.
Principle:
Basic principle of this technique is that when sample undergoes physical transformation such as phase
transitions, more or less heat is needed to flow the sample than reference to maintain both at same
temperature. When more or less heat must flow to sample depend on whether the process is
exothermic or endothermic.
Hence DSC provides qualitative & quantitative data on endothermic (heat absorption) and exothermic
(heat evolution) cause by phase transitions of material.
These phase transitions may be;

Melting


Glass transitions



Crystallization



Oxidations



Other heat related changes.

DSC works in junction with controller & associated software to allow for data acquisition and analysis.

Detection of phase transitions:
As the temperature increases an amorphous solid will become less viscous. At some point molecules
may obtained enough freedom of motion to spontaneously arrange themselves into crystalline form,
this is called Crystallization temperature (Tc), the process is exothermic because less heat is required to
raise temperature of sample and it results a peak in DSC signal.
As, the temperature further increases sample actually reaches its Melting temperature (Tm) this melting
process results in exothermic peak in DSC curve because more/ greater amount of heat is required to
raise the sample temperature.
Instrumentation
DSC is used to study the thermal transitions of polymer sample. The polymer is heated in a device that
looks something like this;
1. Two pans:
There are two pans in the DSC machine;
i. Reference pan: The left one, empty pan is reference pan.
ii. Sample pan: The pan on right side, with polymer sample is sample pan.
2. Heater:
Each pan sits of heaters which are controlled by a computer.
3. Computer:
Computer turns on heater and let them heat the two pans at a specific rate, that is; usually 10 seconds
per minute. Computer make absolutely sure that heating arte stays exactly the same throughout the
experiement.

DSC curve
The result of DSC curve experiment is a curve of heat flux (differential heat flow) verses temperature or
time. The curve can be used to calculate the enthalpy of transitions. This is done by evaluating peak
corresponding to each transition enthalpy of transition can be expressed by following equation;
∆ =
Where,

∆H = Enthalpy of transition


K =Calorimetric constant


A =Area of Curve
Calorimetric constant varies from instrument to instrument. Area under the peak is directly
proportional to heat absorbed or evolved by reaction. While, Height of the peak is directly proportional
to rate of reaction.
Factors affecting DSC curve:
The DSC curves are;
Typical DSC curves A showing exothermic peak and B showing endothermic peak.
2 types of factors affects DSC curves majorly;
1. Instrumental factors: 2. Sample characteristics:
The instrumental factors includes; The sample factors includes;
 Composition of sample  Amount of sample
containers  Nature of sample

Sensitivity of recording system  Particle size
 Recording speed  Heat of reaction
 Furnace heating rate  Thermal conductivity

Heating rates of pan

Determination of heat capacity
Heat capacity:
It is the amount of heat a material can hold. OR, amount of material takes to get a certain temperature
increase is called heat capacity. It is denoted by Cp and is measured in J/ mole.
DSC plot can be used to determine heat capacity. Suppose a polymer is being heated when we start
heating 2 pans, computer will plot difference in heat output of the two heaters against temperature that
is plot of heat absorbed by the polymer against temperature. Plot will look like this at first;
Heat flow is heat (q) supplied per unit time (t).
= =
Whereas, heating rate is temperature increase (∆T) per unit time (t).
= =
∆
By dividing heat flow by heating rate. It tends up with heat supplied divided by temperature increase
that is called heat capacity.
∆ = ∆ =

Schematic DSC Curve
A schematic DSC curve determines several common features;
1. The glass transition temperature (Tg)
It is important characteristic of semicrystalline, amorphous and many polymers.

Below Tg semicrystalline polymers tends to becomehard, brittle.


Above Tg chains easily rotate and becomes softer.

Tg depends on structure, nature and bonding. It takes energy to break bonds. There is more heat flow.
Increase in heat capacity takes energy to break bonds. Transition appears on DSC curves endothermic
process.
2. Crystallization:
Another transition occurs at DSC curve is Tc at this polymer loses its random chain arrangement,
intermolecular bond formation occurs, becomes more ordered (crystals) formation of bond is
exothermic, so in heat flow there is decreased heat flow. This occurs as a big peak in plot of heat flow
versus temperature.
Temperature at highest point in peak is considered polymer’s crystallization temperature or Tc

3. Melting:
It occurs when solid changes into liquid. Molecular bonds absorb energy, began to loose and breaks. It
means little heater under sample pan has to put a lot of heat to polymer. In order to both melt the
crystal and to maintain same rate of temperature. This extra heat flow during melting cause a big dip on
DSC plot as;
Putting it all together transitions we get a graph like this;

Types of DSC:
There are two types of DSC machines available in market;
 Heat flux DSC
 Power compensation DSC



 Heat Flux DSC:
In heat flux DSC sample holders are connected to single heat flow path. Thermocouple sensors are used.
Both the sample and reference pan have the same furnace as a heating source.
 Power compensated DSC:
The sample holders are made up of Aluminum or Platinum metal. Separate sensors for reference
and sample pan is used. Similarly, both the sample and reference pan have separate furnace block.

Pharmaceutical applications
DSC has following applications;
1. Study of matter transitions:
DSC, it is possible to observe small energy changes that occurs as matter transitions that is; from solid
to a liquid crystal.
2. Oxidative stability:
DSC can be used to study the stability to oxidation of samples. Such that are done isothermically by
changing atmosphere conditions. Oxidation is observed as a derivation in base line.
3. Drug analysis:
DSC is usually used in pharmaceuticals and polymer industries.

Cross linking or curing in polymers is observed by DSC plot is an exothermic process.


It is necessary to deliver a drug in amorphous form, it is desirable to process a drug at temperature
below crystallization can occur.


It measures heat loss or gain resulting from physical or chemical change in sample as function of
temperature.


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