DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING

FACULTY OF ENGINEERING

INSTRUMENTAL CHEMICAL ANALYSIS:

BASIC PRINCIPLES AND TECHNIQUES

 2

PREFACE

This manual has been prepared exclusively for the final year undergraduate students and
students doing the master’s programme in a view to outline the basic principles in the
instrumental chemical analysis and techniques. The manual explains the basics in the theory
of instrumentation, their wide classification based on the interactions of the molecules with
the matter and energy, the steps and principles involved in the individual technique and their
applications in various fields. As this work is intended for quick and easy learning, all the
descriptions are kept at concise and simple. This is sufficient for the students to feel
comfortable with the techniques of the instruments. However, appropriate references are
given for advanced and detailed descriptions.

We hope that this could be a good initiative and guidance for the students who, as a part of
their study programme, should pursue with short research works. We strongly advice students
to go through this manual completely before handling the instruments, as this may give some
confidence and familiarisation over the techniques.

The details explained in this manual have been collected from various sources cited at the
reference section and any suggestions or modifications for this manual from the staff
members are welcome.
Good Luck
DR D RAJARATHNAM
 3

INDEX

1. GENERAL INTRODUCTION 5

2. CLASSIFICATION OF THE INSTRUMENTAL TECHNIQUES 6

3. ANALYSIS THROUGH SPECTROSCOPY

3.1 Processes in Spectroscopy 7

3.1.1 Atomic Level

3.1.1a Atomic Absorption Spectroscopy ( AA ) 8
3.1.1b Flame Emission Spectroscopy 9
3.1.1c Plasma Emission Spectroscopy 10
3.1.1d Fluorometry : Atomic Fluorescence 11

3.1.2 Molecular Level

3.1.2a Ultra Violet - Visible Spectroscopy ( UV/VIS ) 11
3.1.2b Infra Red Spectroscopy ( IR ) 12
3.1.2c Raman Spectroscopy ( IR-RAMAN ) 13
3.1.2d Nuclear Magnetic Resonance Spectroscopy ( NMR ) 14
3.1.2e Fluorometry : Molecular Fluorescence 15
3.1.2f Electron Spin Resonance Spectroscopy ( ESR ) 15
3.1.2g Microwave Spectroscopy 16

4. ANALYSIS THROUGH CHROMATOGRAPHY

4.1 LIQUID CHROMATOGRAPHY ( LC/HPLC ) 17
4.1.1 Adsorption Chromatography 17
4.1.2 Partition Chromatography 17
4.1.3 Ion-Exchange Chromatography 17
4.1.4 Size Exclusion Chromatography 18

4.2 GAS CHROMATOGRAPHY( GC ) 19

5. ANALYSIS THROUGH THERMAL ENERGY

5.1 THERMAL ANALYSIS 21
5.1.1 Thermogravimetric Analysis ( TGA ) 21
5.1.2 Thermomechanical Analysis ( TMA ) 22
5.1.3 Differential Thermal Analysis ( DTA ) 22
5.1.4 Differential Scanning Coloriemetry ( DSC ) 23

6. ANALYSIS THROUGH X-RAY TECHNIQUES

6.1 Generation of X-Rays 23
6.2 X-Ray Fluorescence Spectrometry ( XFS ) 24
6.3 X-Ray Photo-emission Spectrometry ( XPS ) 24
6.4 X-Ray Diffractometry ( XRD ) 26
 4

7. ANALYSIS THROUGH MICROSCOPY

7.1 Scanning Electron Microscopy ( SEM ) 27
7.2 Transmission Electron Microscopy ( TEM ) 28
7.3 Scanning Probe Microscopy ( SPM ) 29

8. ANALYSIS THROUGH ELECTRO-CHEMICAL TECHNIQUES

8.1 Polarography 31
8.2 Electrophoresis 34

9. ANALYSIS THROUGH OTHER TECHNIQUES

9.1 Total Organic Carbon Analyzer ( TOC ) 35
9.2 Elemental Analyzer ( CHN Analyzer ) 35
9.3 Polarimetry 35
9.4 UV/Visible Spectropolarimetry 37
9.5 Vibrational Circular Dichroism ( VCD/VLD ) 38
9.6 Mass Spectrometry ( MS ) 39
9.7 Laser Light Scattering System ( Particle Sizing and Analysis ) 40

10. SUMMARY 42

11. REFERENCE 44

---------------------------------------------------------
 5

1. GENERAL INTRODUCTION

The need of the sophisticated analytical instruments and determinations using them is almost
a routine and inevitable nowadays for the modern chemical laboratories. It has been a vast
expanding area of knowledge as the instrument and computer manufacturers are producing
analytical machines, which are in ever-increase of power and scope. Further, all the manual
techniques in the line of the analytical studies had steadily been transferred to the
instrumental techniques. Basically, chemical analysis can be divided into three broad
categories as follows:

QUALITATIVE ANALYSIS : Chemical analysis which just identifies one or more species
present in a sample

QUANTITATIVE ANALYSIS : Chemical analysis which finds out the total amount of the
particular species present in a sample

STRUCTURAL ANALYSIS : Chemical analysis which helps in finding the spatial

arrangement of atoms in a molecule and the presence or
position of certain organic functional groups in a given
compound

These three classes are almost invariably applied to major areas such as Fundamental
Research, Product Development, Product Quality Control, Monitoring & Control of
Pollutants, Medical & Clinical Studies, etc. For all these applications, you can’t imagine of
running a laboratory virtually without modern analytical instruments.

In general, chemical analysis has some basic steps like, Choice of Method, Sampling,
Preliminary Sample Treatment, Separations, Final Measurement and Assessment of Results
(this series is almost equally applicable to the above mentioned three classes). It is with the
first step viz. Choice of Method, one should select the proper instrument to carry out the
analysis with full success. A wrong selection at this point may lead certainly to a meaningless
analysis. Selection of the instrument is such important criteria!

As it is, in order to compete with this type of situation, some basic knowledge of instruments
and analytical techniques are required. This may give the person the ability, with some
confident, to choose and operate a varied range of instruments, which would be much
required at the advanced research laboratories. The following pages will give an insight into
the basics regarding the theory, principles and applications of various analytical instruments.
 6

2. CLASSIFICATION OF THE INSTRUMENTAL TECHNIQUES

In a broad sense the instrumental techniques for the chemical analysis can be sub-divided as
follows. Though this classification doesn’t include few other techniques like radiochemical
analyses and some of the physical methods, it is more than sufficient to start with.

ANALYSIS THROUGH SPECTROSCOPY

ANALYSIS THROUGH CHROMATOGRAPHY

ANALYSIS THROUGH THERMAL ENERGY

INSTRUMENTAL
TECHNIQUES ANALYSIS THROUGH X-RAY TECHNIQUES

ANALYSIS THROUGH MICROSCOPY

ANALYSIS THROUGH ELECTROCHEMICAL

TECHNIQUES

ANALYSIS THROUGH OTHER TECHNIQUES

This classification is based on the interactions of molecules with various forms of energy like
electro-magnetic radiation, heat (thermal energy) and with the matters like electrons. Each
and every technique is having it’s own principle, mode of operation and advantages and
disadvantages.
 7

3. ANALYSIS THROUGH SPECTROSCOPY

3.1 Processes in Spectroscopy

The interaction of the light (electro-magnetic radiation) with a substance and the subsequent
energy transfer ends with three main processes namely:

Absorption : The process by which the energy of the light (in the form of photons) is
transferred to the atom or molecule raising them from the ground state
to an excited state

Fluorescence : The absorbed energy is rapidly lost to the surroundings by collisions

within the system and relax back to the ground state. Sometimes the
energy is not lost in this way but is re-emitted a few milli seconds later,
which we call as fluorescence

Emission : If the substances (atoms or molecules) are heated to high temperatures

(in a flame or in an electric discharge) the electrons are exited to higher
energy levels. Later, they relax to the ground state with the emission of
radiation, the magnitude of which is more or less equivalent to absorbed
energy

Most of the analytical instrumental techniques are based on the light interactions with the
substances and utilise any of the above three associated processes. Substances interact with
light differently at various wavelengths and hence different types of analysis and instruments.
The entire spectra of the light with varying wavelengths can be represented as follows
(Fig.1). Since light is having both electrical and magnetic components, this representation is
referred to as an Electro - Magnetic Spectrum. (For a detailed study on these concepts please
refer to Physical Chemistry text books).

Fig: 1
400nm 700nm
λ (nm)

Gamma X-Rays UV VIS IR Microwaves Radio and

Rays TV Waves

High energy Low energy

High frequency Low frequency
Short wavelength Long wavelength

The following is short comparison between Ultra Violet (UV), Visible (Vis) and Infra Red
(IR) ranges for the energy, frequency and wavelength:

Energy: UV > Vis > IR

Frequency: UV > Vis > IR
Wavelength: UV < Vis < IR
 8

The symbol for the wavelength is “lambda” (λ) and the unit is either nanometer (nm) or
micrometer (or micron, µm). The symbol for frequency is “nu” (µ) and the unit is either
hertz or sec-1. A parameter closely related to frequency is the wave number, which has the
symbol “nu bar” (∨) and the unit is cm-1.

There are two levels by which the substances can interact with the light as, atomic level and
molecular level and hence the corresponding techniques:

3.1.1 Atomic Level

3.1.1a Atomic Absorption Spectroscopy (AA):
Principles: The analyte sample is vaporized by aspiration of solution into a flame or
evaporation from electrically heated surface. At this condition where the individual atoms co-
exist, a beam of light is passed through them. The atoms will absorb in the visible and
ultraviolet region resulting in changes in electronic structure (excited state). So, the resultant
light beam coming out of the sample will be missing the light in the corresponding wave
length, which is a measure of the characteristics of the sample.

Instrumentation: Sources emitting radiation characteristic of element of interest (hollow -

cathode lamp), flame or electrically heated furnace, monochromator, detector
(photomultiplier) and recorder. The following is the simplified outline of the
instrumentation:

Fig: 2

Flame Filter
Monochromator
Light Source

Detector

Spray Chamber

Fuel
Recorder

Oxidant

Aspiration Tube

Sample
 9

Applications: This is the most widely used technique for the quantitative determination of
metals at trace levels (0.1 to 100ppm), which present in various materials. It utilizes Beer -
Lambert Law for the analysis and a standard curve is obtained by plotting absorbance vs
concentration of the samples taken. The usual procedure is to prepare a series of standard
solutions over a concentration range suitable for the sample to be analysed. Then, the
standards and the samples are separately aspirated into the flame, and the absorbances read
from the instrument. The plot will give the useful linear range and the concentrations of the
samples can be found out from the plot.

Disadvantages: Sample must be in solution or at least volatile. Individual source lamp and
filters needed for each element, since, each and every metal has its own characteristic
absorption.

3.1.1b Flame Emission Spectroscopy:

Principles: This is simply called as Flame Photometry. It is the technique which measures
the atoms excited by a flame and not by light source as in the atomic absorption case. After
excitation, atoms will readily lose the gained energy and revert back to the ground state and
the emission occurs. It is that emission that actually we measure. The wavelengths of the
emitted light will almost be similar as those that were absorbed in the atomic absorption,
since exactly the same energy transitions occur, except in the order of reverse!

Instrumentation: A simple flame photometer consists of burner, nebulizer, monochromator,

detector and recorder. The following is the simplified figure:

Fig: 3

Flame

Monochromator Detector

Slit

Fuel

Air Nebulizer

Aspiration Tube

Sample
 10

Applications: It is used exclusively in the quantitative determination of metals in solution,

especially alkali and alkaline earth in the given samples. The principle is like that described
for atomic absorption. Qualitative determination is also possible as each element emits its
own characteristic line spectrum.

Disadvantages: Intensity of emission is very sensitive to changes in flame temperature.

Usually, spectral interference and self-absorption are also encountered which affects the
precision of the measurement. Further, a linear plot of absorbance vs concentration is not
always expected.

3.1.1c Plasma Emission Spectroscopy:

Principles: Mostly referred as Inductively Coupled Plasma (ICP) Emission , is essentially an
atomic emission technique, most closely related to the preceded flame photometry except in
that the atoms and ions present in the sample are excited in a high temperature gas plasma.
Since the plasma provides very high temperature and hence the energy, almost all the atoms
present in the sample can be excited with this technique ending up with high efficiency (a
hotter source increases both atomization efficiency and excitation efficiency). Thus, the
emissions from the atoms would be more intense and even very small concentrations of metal
ions can be detected and accurately measured.

Instrumentation: This is basically an emission spectrometer comprising nebulizer, RF coil,

ICP Source (Argon plasma), monochromator, detector and recorder.

Fig: 4
Slit

Plasma Jet
Monochromator Detector

RF Coil

Quartz Tube

Tesla Coil Recorder

Flow Spoiler

Nebulizer

Argon Gas

Aspirator

Sample
 11

A plasma source or jet is a flame-like system of ionized, very hot flowing argon gas. At high
temperatures (≈ 6000 K) a gas such as argon will contain a high proportion of ions and free
electrons constituting a plasma.(This ionisation is initiated by “Tesla” coil). Additional
energy may be supplied to the electrons in the plasma by the application of an external
electromagnetic field, through RF coil. By collisions between the electrons and other species
in the plasma this additional energy is uniformly distributed. As the collisions increase, the
energy transfer becomes more efficient, which leads to a substantial temperature
enhancement to a range of 8000 - 10000 K. It is the temperature at which the samples are
introduced and analysed.

Applications: The same like to that of atomic emission spectroscopy but it covers very
widespread for both qualitative and quantitative analysis of metals and some non-metals too,
at trace levels. Because of the high temperature and homogeneity of the source, it offers
better signal stability and hence the analytical precision.
The technique when utilises the optical emission detector then it is termed as Inductively
Coupled Plasma – Optical Emission Spectrometer (ICP-OES) and if it utilises a mass
spectrometer (refer section 9.4 ) as detector then it is termed as Inductively Coupled Plasma
– Mass Spectrometer (ICP-MS ).

Disadvantages: Samples require dissolution before analysis. Instrumentation is complex and

requires high operator’s skill and is very expensive.

3.1.1d Fluorometry: Atomic Fluorescence:

This technique is not widely used though its counterpart - the molecular fluorescence is
applied well to the analytical studies till date. The principle of atomic fluorescence is that
when atoms are elevated to higher energy levels, they sometimes return to the ground state
through a pathway, which has several intermediate electronic states, before reaching to the
actual ground state. Such series of fall through the electronic levels accompany by light
emission - which is atomic fluorescence. The intensity of this emitted light is measured at
right angles to the incident light and related to concentration.

3.1.2 Molecular Level

3.1.2a Ultra Violet - Visible Spectroscopy (UV/VIS):
Principles: It involves the absorption of electromagnetic radiation by the substances in the
visible and ultraviolet regions of the spectrum. This will result in changes in the electronic
structure of ions and molecules.

Instrumentation: It consists of a dual light source viz., tungsten lamp for visual range
measurements and deutrium lamp for measurements at ultra - violet regions, grating
monochromator, photodetector, mirrors and glass or quartz cells.
NOTE : For measurements to be made under visible region both glass and quartz cells
can be used. For the measurements under ultra-violet region, only quartz cell should be
used, since, glass cells absorb ultra-violet rays.

There are two types of instruments for this technique as single beam and double beam
spectrophotometers. However, nowadays, double beam spectrophotometers are widely used
and following is the outline of the instrument:
 12

Fig: 5

Reference Beam Reference Cell

Detector
Light From
Monochromator

Sample Beam Sample Cell

Sample Compartment

Applications: It is the most widely used technique for quantitative trace analysis, for this
Beer-Lambert law is applied. Sometimes it is used in conjunction with other techniques in the
identification and structural analysis of organic materials. For qualitative analysis it provides
a valuable information, the so-called “molecular absorption spectrum” is obtained, which
exactly tells the nature of the compound - since no two compounds can have the same
absorption and hence the spectra.

Disadvantages: Samples should be in solution. Mixture of substances poses difficult to

analyse and requires prior separation. Interference sometime makes the measurement
difficult, but these disturbances are quite common with these types of techniques.

3.1.2b Infra Red Spectroscopy (IR):

Principles: It involves the absorption of electromagnetic radiation in the infrared region of
the spectrum which results in changes in the vibrational energy of molecule. Since, usually
all molecules will be having vibrations in the form of stretching, bending, etc., the absorbed
energy will be utilised in changing the energy levels associated with these. It is a valuable
and formidable tool in identifying compounds.

Instrumentation: Basically it is a double beam spectrophotometer comprising IR source

(usually a red hot ceramic material), grating monochromator, thermocouple detector, cells
made of either sodium chloride or potassium bromide materials, etc.,. In this process the light
is dispersed by the monochromator. But, this type of basic design for IR measurements has
been outdated. Instead a newer technique termed Fourier Transform Infra Red (FTIR)
spectroscopy becoming increased importance. This technique utilises a single beam of
undispersed light and has the instrumentation essentially similar to the previous one.
 13

In FTIR, the undispersed light beam is passed through the sample and the absorbances at all
wavelengths are received at the detector simultaneously. A computerized mathematical
manipulation (known as “Fourier Transform” ) is performed on this data, to obtain absorption
data for each and every wavelength. To perform this type of calculations interference of light
pattern is required for which the FTIR instrumentation contains two mirrors, one fixed and
one moveable with a beam splitter in between them. Before running with the sample you
should run the instrument with a reference or a blank. The following is the simplified version
of the instrument:

Fig: 6

Detector

Sample

Beam Splitter
Light From IR Source Movable Mirror

Fixed Mirror

Applications: It finds extensive use in the identification and structural analysis of organic
compounds, natural products, polymers, etc.,. The presence of particular functional group in a
given organic compound can be identified. Since, each and every functional group has its
own vibrational energy, the IR spectra can be seen as their fingerprints.

Disadvantages: Samples containing mixture of substances can not be analysed. Since the
sample holders and beam splitter, are made of moisture sensitive materials like sodium
chloride or potassium bromide (KBr), special cells are required for aqueous samples (KRS-5,
ZnSe).

3.1.2c Raman Spectroscopy (IR-Raman):

This technique is actually an extension to FTIR and works in the near IR range. It is an
emission technique, whereby a laser beam is directed to the sample and the scattered
radiation is collected. Most of the scattered radiation has the same wavenumber as that of the
incident laser beam, however a fraction will be having a different wavenumber. This is the
Raman signal and it provides information on functional groups, which give weak or no
signals in the infrared region. Raman spectroscopy finds applications in identifying organic
compounds containing non-polar bonds such as carbon - carbon double bonds or aromatic
rings.
 14

3.1.2d Nuclear Magnetic Resonance Spectroscopy (NMR):

Principles: The techniques which we have seen above utilises the absorption of light in the
visible, ultra-violet and infra red ranges involving changes in electronic states as well as in
molecular vibrational and rotational levels. In NMR, the absorption by substances occurs in
the radio frequency region of the electro-magnetic spectrum resulting in changes in the
orientation of the spinning nuclei. This is observed by applying a magnetic field. It is a well
known fact that the nuclei of the atoms bonded to each other in molecules spin on an axis like
a top. Since nuclei are positively charged, this spin will create a small magnetic field around
it. If an external magnetic field, suppose, applied to these nuclei, then the spin of the nuclei
will align to the magnetic field deviating from the original axis of spin. In this situation if
radio waves are applied to the system, the nuclei will absorb this energy and re-align back to
their original axis of spin. This will give the information regarding the nature of the
compounds and the presence of various functional groups and their environment.
Since this technique is mostly measures the spinning of the hydrogen nuclei (almost all the
organic compounds contain hydrogen atoms!), it is sometimes referred as Proton Magnetic
Resonance (PMR ) spectroscopy.

Instrumentation: The instrumentation for this technique include powerful magnet, radio-
frequency signal generator, amplifier, detector, etc.,. The following is the outline of the
instrument:

Fig: 7
RF Transmitter RF Amplifier Detector

Magnet Magnet

Sweep Generator Sample Tube

Applications: The application lies mostly in the identification and structural analysis of
organic compounds and thus, it is mostly a tool for qualitative analysis. It gives valuable
information regarding the position of the functional groups in a molecule and provides
distinguished spectra for the isomer. Much precise information on the structure of the
compounds can be obtained using the same technique with other magnetic nuclei like C 13,
O17. The instrumentation being the same except that the sweep by the magnetic field is varied.

Disadvantages: Very expensive and the instrumentation is complex and needs exceptional
skills to operate. Its sensitivity ranges from moderate to poor, however, can get clear
information using C13 or O17 NMR. The usage of the solvents is limited and in most of the
situations deuterated solvents are required.
 15

3.1.2e Fluorometry: Molecular Fluorescence:

Principles: This technique utilises the phenomeno of molecular fluorescence, the theory
behind this is exactly the same that has been discussed under atomic fluorescence. Here, most
often the irradiating light is UV light and the emitted light is visible light.

Instrumentation: The instrumental set-up is a simple one which comprises of a UV source,

two monochromators, detector and recorder. The fluorescence exhibited by the sample is
measured at right angles to the incident beam. The following is the basic set-up:

Fig: 8

UV Source Absorption Sample Transmitted

Monochromator Light

Fluorescence
Monochromator

Detector

Recorder

Applications: The applications of this technique is limited and it offers quantitative

estimations of those compounds like benzene and fused benzene ring systems. Inorganic
metals can also be analysed by the ability of them to form complexes with the ligands. It
finds uses in the analysis of foods for vitamin content, since vitamins like riboflavin, niacin,
etc., exhibit fluorescence, it is a good tool for this area. Since only limited compounds show
the fluorescence, this technique is relatively free of any interference and is very sensitive.

Disadvantages: The application is very limited, as only a few of the substances exhibit
flourescence.

3.1.2f Electron Spin Resonance Spectroscopy (ESR):

The basic principle of electron spin resonance spectroscopy is that, electrons always have a
spin and thus have a magnetic moment. Thus, the magnetic resonance theory applies to
 16

electrons too like that of nuclei, as in NMR!. Especially this technique is of high value when
it comes to the compounds which contain odd electrons, that is, those substances which have
paramagnetic behaviour (if electrons are paired as in bonded orbital then their mutual
spinning will cancel each other and there will be no response for the applied magnetic field,
whereas, if it is unpaired then it can align with the applied magnetic field and the feasibility
of getting ESR spectra is higher). Thus, the principle and the instrumentation are much
similar to that of NMR technique. It is also referred as, Electron Magnetic Resonance
(EMR) or Electron Paramagnetic Resonance (EPR) spectroscopy.

It is mostly used as a potential technique to study the formation and lifetime of free radicals,
which are the major intermediates in most of the organic reactions. Another important field of
application of ESR is in the estimation of trace amounts of paramagnetic ions, particularly in
biological works like, Mn2+, Mg2+, etc.

3.1.2g Microwave Spectroscopy:

This technique is actually an extension to IR spectroscopy. Microwave region lies at the far
infra-red region of the electromagnetic spectrum and its absorption by molecules give rise to
changes in the rotational energies of the molecules. Remember in IR spectroscopy, the
molecules are subjected for changes in vibrational energies; the energy required for making
changes at rotational levels is lesser than that for vibrational levels. Though the principles are
same to that of IR, the instrumentation is slightly different in that it requires samples in
gaseous state for the analysis.
Its applications are limited to smaller and simpler molecules since, larger molecules will have
interactions between the rotational energy levels within the molecule through various bonds
they have. In certain cases this technique can be considered as a good alternate to IR
spectroscopy. Besides qualitative analysis, this technique can be applied for conformational
analysis of simpler compounds (study of stereo chemistry of the compounds).

4. ANALYSIS THROUGH CHROMATOGRAPHY

The technique through which the chemical components present in complex mixtures are
separated, identified and determined is termed as chromatography. This technique is also
widely used like spectroscopy and is a very powerful tool. This technique while provides a
way for analytical purposes is on the other hand used for preparative methods too.
Compounds of high-grade purity can be obtained by this way. It is very difficult to define the
term “Chromatography” as a whole, as many, many systems and techniques are embedded
into this term. However, it can be simply defined as follows:
“It is the technique in which the components of a mixture are separated based upon the
rates at which they are carried or moved through a stationary phase by a gaseous or liquid
mobile phase”.
Based on the mobile phase this technique can be simply classified in to two categories as:
Liquid Chromatography and Gas Chromatography. The so called “column” which holds the
stationary phase (which in the form of small particles of the diameter of the order in microns)
plays unique role in these processes. Usually silica is the base material for producing this
phase.
 17

4.1 LIQUID CHROMATOGRAPHY (LC/HPLC)

Principles: Early liquid chromatography was carried out in long glass columns with wide
diameter. The diameters of the stacked particles inside the column were of the order of 150-
200 microns range. Even then, the flow rates (eluent time) of the mobile phase with the
analyte were very slow and separation times were long - often several hours!. With the
advent of latest technology the particle diameters were reduced as small as to 10 microns
with replacement of glass columns with steel ones. The speed of the flow rates were
improved by applying high pressure to the column using pumps and hence the performance
was improved. This development led the instrument to be mostly called as “High-
Performance Liquid Chromatography” or “High-Pressure Liquid Chromatography”
(HPLC). Though HPLC retains major of the credits to the analytical side, the earlier one of
simple Liquid Chromatography still finds applications in the preparative purposes.
The HPLC technique can be divided into four main categories depending on the nature of the
processes that occur at the columns as follows:

4.1.1 High-Performance Adsorption Chromatography: Here as the name implies the analyte
species (components to be analysed) are adsorbed onto the surface of a polar packing. The
stationary phase consists of finely divided solid particles packed inside a steel tube. If the
component mixture is eluted through this tube with the mobile phase, different components
present in the mixture adsorb to different degrees of strength and they become separated as
the mobile phase moves steadily through the column. The nature of the adsorption involves
the interaction of polar molecules with a very polar solid stationary phase. The stationary
phase could be silica gel or alumina. This method is extensively used for the separations of
relatively nonpolar, water-insoluble organic compounds. One particular application is in
resolving isomeric mixtures such as meta-, and para-substituted benzene derivatives.

4.1.2 High-Performance Partition Chromatography: It is the most widely used of all liquid
chromatographic procedures. Here the components present in the analyte mixture distribute
(or partition) themselves between the mobile phase and stationary phase as the mobile phase
moves through the column. The stationary phase actually consists of a thin liquid film either
adsorbed or chemically bonded to the surface of finely divided solid particles. Of these the
latter is considered more important and has a distinct stability advantage. It is not removed
from the solid phase either by reaction or by heat and hence it is more popular. It finds wide
applications in various fields which include, pharmaceuticals, biochemicals, food products,
industrial chemicals, pollutants, forensic chemistry, clinical medicine, etc.

4.1.3 High-Performance Ion-Exchange Chromatography: It is the method used to separate

mixtures of ions, both inorganic and organic in nature. It finds its application mostly in
protein separations. Here, the stationary phase consists of very small polymer resin “beads”
which have many ionic bonding sites on their surface, termed as Ion Exchange Resins. This
resin can be either an anion exchange resin, which possesses positively charged sites to
attract negative ions, or a cation exchange resin, which possesses negatively charge sites to
attract positive ions. If the analyte mixture which contains mixture of ions is introduced into
the column packed with suitable ion-exchange resin, selected ions will be attached or bonded
on to the resin, thus being separated from other species that do not bond. Later, these attached
ions can be dislodged from the column by repeated elution with a solution that contains an
ion that competes for the charged groups on the resin surface, in other words, which has high
affinity for the charged sites on the resin than the analyte ions. Thus the analyte ions get
exchanged and separated from the column.
 18

4.1.4 High Performance Size Exclusion Chromatography: It is a technique for separating

dissolved species on the basis of their size. It is particularly applicable to high-molecular-
weight species like oligomers and polymers to find out their relative size and molecular
weight distribution. Here, the stationary phase is polymer resin, which contains small pores.
So if the components to be separated are passed through the column the small sized particles
can easily enter into these pores and their mobility is retarded. Whereas the large sized
particles, which can’t enter into these pores can come out of the column fast and elude first.
Thus the separation of various sized particles is possible through variations in the elution
time. It is classified into two categories based on the nature of the columns and their packing
as:

Gel Filtration Chromatography - which uses hydrophilic packing to separate polar species
and uses mostly aqueous mobile phases. This technique is mostly used to identify the
molecular weights of large sized proteins.
Gel Permeation Chromatography - which uses hydrophobic packing to separate nonpolar
species and uses nonpolar organic solvents. This technique is used to identify the molecular
weights of polymers.

Instrumentation: The basic HPLC system consists of a solvent (mobile phase) reservoir,
pump, injection device, column and detector. The pump draws the mobile phase from the
reservoir and pumps it through the column. At the head of the column is the injection device,
which introduces the sample to the column. On the end of the column (effluent end), a
detector is positioned. In most of the cases the detector used is UV absorption detector. In the
case of analytical studies, after the detection the eluents are collected in waste bottles. In the
case of preparative studies the eluents are fractionally collected for further studies.
Most of the HPLC design will be the same as described for all the four main groups
previously described. However, there can be differences in selecting the specific detectors for
particular type of analysis, say for example, with ion-exchange chromatography, detectors
commonly used are conductivity detectors for obvious reasons. The following is the most
generalised outlay of the HPLC system: Note: column depiction is not to size

Fig: 9

Pump Injection Point

Detector

Recorder

Collector
Mobile Phase Column
 19

Disadvantages: Column performance is very sensitive, which depends on the method of

packing. Further, no universal and sensitive detection system is available.

4.2 GAS CHROMATOGRAPHY (GC)

Principles: Here an inert carrier gas (Helium or Nitrogen) acts as the mobile phase. This will
carry the components of analyte mixture and elutes through the column. The column usually
contains an immobilized stationary phase. The Gas Chromatography can be categorised
depending on the nature of the stationary phase that involved as follows:
Gas Solid Chromatography (GSC) - here the stationary phase is a solid which has a large
surface area at which adsorption of components of the analyte takes place. The separation is
possible based on the differences in the adsorption power and its equilibria between the
components and the solid surface of the stationary phase. The application of this method is
limited and is mostly used in the separation of the low-molecular-weight gaseous species like
carbon monoxide, oxygen, nitrogen and hydrocarbons.
Gas Liquid Chromatography (GLC) - this is the most important and widely used methods for
separating and determining the chemical components of complex mixtures. Here the
stationary phase is a liquid that is immobilized on the surface of a solid support by adsorption
or by chemical bonding. The separation of the mixture into individual components is by
distribution ratio (partition) of these anayte components between the gaseous phase and the
immobilized liquid phase. Because of its wide applications most of the GCs are configured
for the GLC technique.

Instrumentation: The instrumentation for GC is slightly different from that of HPLC in that
the injection port, column and detector are to be heated to a pre-specified temperature. Since
the mobile phase here is a gas (carrier gas) the components present in the analyte mixture
should be vaporised, so that it can be effectively carried into the column. The basic
instrumentation for GC includes a carrier gas cylinder with regulator, a flow controller for the
gas, an injection port for introducing the sample, the column, the detector and the recorder.
An outlay is as follows:

Fig:10

Pressure Regulator Injection Port Column Oven Column Detector

Gas Flow

Carrier Gas Recorder

In the above illustration the injection port, detector and column oven are hot zones. The
success of this technique requires the appropriate selection of the column for the particular
 20

analysis, since it is the heart of the process, and the temperature conditions at which the
column to be maintained throughout the analysis. Basically the column for the GC purposes
are classified as analytical columns and preparative columns. The analytical columns are of
two types: packed column and open-tubular or capillary column. Both differ in the way the
stationary phases are stacked inside.

In the instrumentation of GC detectors play unique role. There are a number of detectors,
which vary in design, sensitivity and selectivity. Detectors in GC are designed to generate an
electronic signal when a gas other than the carrier gas elutes from the column. Few examples
and applications of the detectors are:
Thermal Conductivity Detector (TCD) - this operates on the principle that gases eluting from
the column have thermal conductivity different from that of the carrier gas. It is the universal
detector ( detects everything ) and is non-destructive and hence used with preparative GC,
but less sensitive than other detectors.
Flame Ionization Detector (FID) - it is one of the important detectors. Here the column
effluent is passed into a hydrogen flame where the flammable components are burned. In this
process a fraction of the molecules gets fragmented into charged species as positive and
negative. While positively charged ions are drawn to a collector, negatively charged ions are
attracted to positively charged burner head, this creates an electric circuit and the signal is
amplified. The FID detector is very sensitive, but destroys the sample by burning. It only
detects organic substances that burn and fragment in a hydrogen flame. Hence its usage is
restricted for preparative GC and for inorganic substances which do not burn.
Electron Capture Detector (ECD) - this is another type of ionization detector which utilises
the beta emissions of a radioactive source, often nickel-63, to cause the ionization of the
carrier gas molecules, thus generating electrons which constitute an electrical current. This
detector is used for environmental and bio-medical applications. It is especially useful for
large halogenated hydrocarbons and hence in the analysis of halogenated pesticide residues
found in environmental and bio-medical samples. It is extremely sensitive. It does not destroy
the sample and thus may be used for the preparative work.
Nitrogen/Phosphorus Detector (NPD) - the design of the detector is same to that of the FID
detector except that a bead of alkali metal salt is positioned just above the flame. It is also
known as Thermionic Detector. It is useful for the phosphorus and nitrogen containing
pesticides, the organophosphates and carbamates. The sensitivity for these compounds are
very high since, the fragmentation of the other organic compounds are minimized.
Flame Photometric Detector (FPD) - here a flame photometer is incorporated into the
instrument. The principle is that the sulfur or phosphorus compounds burn in the hydrogen
flame and produce light emitting species. This detector is specific for organic compounds
containing sulphur or phosphorus. It is very selective and very sensitive.
Electrolytic Conductivity Detector (ECD Hall) - this otherwise known as Hall detector,
converts the eluting gaseous components into ions in liquid solution and then measures the
electrolytic conductivity of the solution in a conductivity cell. The conversion to ions is done
by chemically oxidizing or reducing the components with a “reaction gas” in a small reaction
chamber. This detector is used in the analysis of organic halides. This detector has excellent
sensitivity and selectivity, but is a destructive detector.

The recent development with the GC instrumentation is that, this has been adopted as the
“hyphenated technique”, ie. by combining the GC with the other analytical techniques like
Infra Red Spectrometry (Gas Chromatography-InfraRed spectrometry, GC-IR) and Mass
Spectrometry (Gas Chromatography- Mass Spectrometry, GC-MS). These are very
 21

powerful tools for qualitative analysis as very accurate and precise information like mass or
IR spectrum of the individual sample components are readily obtained as they elute from the
GC column. It saves time and reduces the steps involved for a component to be separated and
analysed.

Disadvantages: Samples must be volatile and thermally stable below about 4000 C. No single
universal detector is available and most commonly used detectors are non-selective. One
should take much care in the analytical steps starting from the selection of the column, the
detector and must define the temperatures of all the three ports viz., injection port, column
oven and detector. An improper calculation on these will lead to insensitive results.

5. ANALYSIS THROUGH THERMAL ENERGY

5.1 THERMAL ANALYSIS
The technique of thermal analysis actually comprises of a series of methods, which detect the
changes in the physical and mechanical properties of the given substance by the application
of heat or thermal energy. The physical properties include mass, temperature, enthalpy,
dimension, dynamic characteristics and others. In other words it detects the dependency of
physical properties with the temperature and tells the condition of the substances. It finds its
application in finding the purity, integrity, crystallinity and thermal stability of the chemical
substances under study. Sometimes it is used in the determination of the composition of
complex mixtures. This technique has been adopted as testing standard in quality control in
the production field, process control and material inspection. It is applied in wide fields,
including, polymer, glass, ceramics, metals, explosives, semiconductors, medicines and
foods.
The following are the popular methods under this technique:

5.1.1 Thermogravimetric Analysis (TGA):

Simply it is also called as thermogravimetry (TG). Here the change in sample weight is
measured while the sample is heated or cooled at a constant rate. This technique is effective
for quantitative analysis of thermal reactions that are accompanied by mass changes, such as
evaporation, decomposition, gas absorption, desorption and dehydration. The following is the
simplified diagram for the instrumentation:

Fig: 11

Mircro Balance Recorder

Reference Pan

Furnace Sample Pan

 22

The micro-balance plays a significant role here. During the process of measurement, the
change in sample mass affects the equilibrium of the balance. This imbalance is fed back to a
force coil, which generates additional electromagnetic force to recover equilibrium. The
amount of additional electromagnetic force is proportional to the mass change. During the
heating process the temperature may go as high as 10000 C inside the furnace.

5.1.2 Thermomechanical Analysis (TMA):

Thermomechanical analysis is the measurement of a material’s behaviour, ie. expansion or
contraction, when temperature or a load is applied. A scan of dimensional changes related to
temperature or load provides valuable information about the sample’s mechanical properties.
One of the most important applications of TMA is the characterization of composite and
laminate materials, through the study of glass transition temperature and the expansion
coefficient. Another application is in the quantitative measurement of extension and
contraction observed in textile fibres, thin films and similar materials.

5.1.3 Differential Thermal Analysis (DTA) :

This technique of DTA measures the temperature difference between a sample and a
reference material as a function of temperature as they are heated or cooled or kept at a
constant temperature (isothermal). In actual practice, the sample and reference material are
simultaneously heated or cooled at a constant rate. Reaction or transition temperatures are
then measured as a function of the temperature difference between the sample and reference.
It provides vital information of the materials regarding their endothermic and exothermic
behaviour at high temperatures. It finds most of its applications in analysing and
characterising clay materials, ceramic, ores, etc. The following is the simple outline of the
instrument:

Fig: 12

Furnace
Sample Pan Reference Pan

Thermocouple

Detector

Signal Recorder
Amplifier
 23

5.1.4 Differential Scanning Caloriemeter (DSC):

This technique is more or less similar to DTA except that it measures the amount of heat
absorbed or released by a sample as it is heated or cooled or kept at constant temperature
(isothermal). In actual practice the sample and reference material are simultaneously heated
or cooled at a constant rate. The difference in temperature between them is proportional to
the difference in heat flow (from the heating source ie. furnace), between the two materials. It
is the well-suited technique in the detection and further studies of liquid crystals. This
technique is applied to most of the polymers in evaluating the curing process of the thermoset
materials as well as in determining the heat of melting and melting point of thermoplastic
polymers. Through the adjunct process of isothermal crystallization it provides information
regarding the molecular weight and structural differences between very similar materials. The
instrumentation is exactly similar to that of DTA except for the difference in the
manipulation of the results.

6. ANALYSIS THROUGH X-RAY TECHNIQUES

6.1 Generation of X-Rays: When metals, like copper, molybdenum, tungsten, etc., are
bombarded directly with a stream of high energy electrons or radioactive particles, X-Rays
(of wavelengths of order 0.1-100Å) are emitted because of the transitions involving K-Shell
and L-Shell electrons. This can be simply expressed as follows:

A cathode in the form of a metal wire when electrically heated gives off electrons. If a
positive voltage, in the form of an anode (target comprised of the metals mentioned above), is
placed near these electrons, the electrons are accelerated toward the anode. Upon striking the
anode, the electrons transfer their energy to the metallic surface, which then gives off X-ray
radiation. For technical reasons these X-rays are sometimes referred as primary X-rays. The
following is the schematic diagram for the process:

Fig: 13

Electrons accelerating towards anode

Vacuum

Hot Cathode Anode

X-Rays

Window

Note: The wavelength of the emitted X-ray is characteristic of the element being
bombarded. Hence with some modifications this process can be used as a tool for
qualitative and quantitative elemental analysis by measuring the wavelength and emission
intensity of the X-rays respectively. This forms the basis for Electron Probe Microanalysis!
(Ref: Sec 7.1)
 24

6.2 X-Ray Fluorescence Spectrometry (XFS):

Principles: When a sample is placed in a beam of primary X-rays, part of it will be absorbed
and the atoms get excited, by the ejection of electrons present in K and L shells. While
relaxing they re-emit X-rays of characteristic wavelength. This re-emitted X-rays are called
secondary or fluorescent X-rays and hence the name for this technique. Since, the
wavelength of the fluorescence is characteristic of the element being excited, measurement of
the wavelength and intensity enables to carry out the qualitative and quantitative analyses.

Instrumentation: The instrumentation is somewhat complicated which accompany, source

for primary X-rays, collimators, analyzing crystal and detector.

Applications: It is one of the non-destructive methods in the elemental analysis of solid or

liquid samples for major and minor constituents. Most of the elements in the periodic table,
both metals and nonmetals, respond to this technique. Detection limit is between 10 to 100
ppm. One of the significant use of this method is in the medical field in identifying and
determining the sulfur in protein.

Fig: 14

Sample
Detector
Collimators

Source

Analysing Crystal

Disadvantages: The sensitivity gets affected for elements with lower atomic numbers,
particularly elements with atomic number lower than 15 are difficult to analyze. The
sensitivity is also limited by matrix absorption, secondary fluorescence and scattering of the
particles. Instruments are often large, complicated and costly.

6.3 X-Ray Photo-emission Spectrometry (XPS) :

Principles: When a primary X-ray beam of precisely known energy impinges on sample
atoms, inner shell electrons are ejected and the energy of the ejected electrons is measured.
The difference in the energy of the impinging X-ray and the ejected electrons gives the
binding energy (Eb) of the electron to the atom. Since, this binding energy of the emitted
electron depends on the energy of the electronic orbit and the element it can be used to
identify the element involved. Further, the chemical form or environment of the atom affects
the binding energy to a considerable extent to give rise to some chemical shift, which can be
 25

used to identify the valence of the atom and its exact chemical form. This technique is mostly
referred as Electron Spectroscopy for Chemical Analysis (ESCA), since it involves the
study of the ejected electrons.

An associated process with this method is that when the electron is ejected from the inner
orbital a vacancy is left with. Hence, another electron from the outer orbits may fall to fill the
vacancy and by doing so emits X-ray fluorescence. The energy of this X-ray fluorescence is
sometimes transferred to a second electron to make it to be ejected. This second electron thus
emitted is termed as Auger electron and the method Auger Spectroscopy (after French
Physicist Pierre Auger). Its applications are more or less similar to ESCA and both the
methods are used in conjunction since in both cases the energies involved are similar.

Instrumentation: It consists of a radiation source for primary X-rays, monochromator, the

energy analyzer (to resolve the electrons generated from the samples by energy) and detector
to measure the intensity of the resolved electrons. The analysis is done in high vacuum.

Fig: 15

Sample Inlet
System

Electron
Source Sample Energy
Analyser

Pumping

Detector

Applications: It is mainly used for surface analysis, especially in the qualitative identification
of the elements in a sample. Based on the chemical shifts, the chemical environment around
the atoms can also be estimated. This measurement is useful in determining the valence states
of the atoms present in various moieties in a sample. Quantitative measurements can be made
by determining the intensity of the ESCA lines of each element.

Disadvantages: High vacuum is necessary for the system to avoid the low energy electrons to
be collided with other impurities, which may result in low sensitivity. It is not possible to
 26

detect the impurities at the ppm or ppb levels. The whole instrumentation is highly
complicated.

6.4 X-Ray Diffractometry (XRD):

Principles: In this technique the monochromatic primary X-rays are made to fall on the
sample substance under study. Because of its wave nature, like light waves, it gets diffracted
to a certain angle. This angle of diffraction, which differs from that of the incident beam, will
give the information regarding the crystal nature of the substance. The wavelength of the X-
rays can be varied for the application by using a grating plate.

Instrumentation: It consists of X-ray tube for the source, monochromator and a rotating
detector.

Fig: 16

Sample

Source
(X-ray Tube)

Detector
Movement of Detector

Applications: The diffraction of X-rays is a good tool to study the nature of the crystalline
substances. In crystals the ions or molecules are arranged in a well-defined positions in
planes in three dimensions. The impinging X-rays are reflected by each crystal plane. Since
the spacing between the atoms and hence the planes can’t be same or identical for any two
chemical substances, this technique provides vital information regarding the arrangement of
atoms and the spacing in between them and also to find out the chemical compositions of
crystalline substances. The sample under study can be of either a thin layer of crystal or in a
powder form. Since, the power of a diffracted beam is dependent on the quantity of the
corresponding crystalline substance, it is also possible to carry out quantitative
determinations.

7 ANALYSIS THROUGH MICROSCOPY

The techniques described here are not for the simple, ordinary optical microscopes. Optical
microscopes are used for the examination and characterization of matter at enhanced
magnification using visible light. The information is obtained by light transmitted or reflected
from matter. The methods explained below are about the advanced microanalysis of surfaces
to nano-scale levels through specialised techniques of the kind of their own.
 27

7.1 Scanning Electron Microscopy (SEM):

Principles: In this technique, an electron beam is focused onto the sample surface kept in a
vacuum by electro-magnetic lenses ( since electron possesses dual nature with properties of
both particle and wave an electron beam can be focused or condensed like an ordinary light! )
The beam is then rastered or scanned over the surface of the sample. The scattered electron
from the sample is then fed to the detector and then to a cathode ray tube through an
amplifier, where the images are formed, which gives the information on the surface of the
sample.

Instrumentation: The instrumentation is very large and comprises of heated filament as

source of electron beam, condenser lenses, aperture, evacuated chamber for placing the
sample, electron detector, amplifier, CRT with image forming electronics, etc.

Fig: 17

Electron gun

Condenser lenses

Objective lens

scanning coils

Detector

Amplifier CRT
Sample
 28

Applications: Scanning electron microscopy has been applied to the surface studies of metals,
ceramics, polymers, composites and biological materials for both topography as well as
compositional analysis. An extension (or sometimes conjunction to SEM) of this technique is
Electron Probe Micro Analysis (EPMA), where the emission of X-rays, from the sample
surface, is studied upon exposure to a beam of high energy electrons. Depending on the type
of detectors used this method is classified in to two as: Energy Dispersive Spectrometry
(EDS) and Wavelength Dispersive Spectrometry (WDS). This technique is used extensively
in the analysis of metallic and ceramic inclusions, inclusions in polymeric materials,
diffusion profiles in electronic components.

Disadvantages: The instrumentation is complicated and needs high vacuum for the optimum
performance.

7.2 Transmission Electron Microscopy (TEM):

Principles: In this technique, a beam of high-energy electrons ( typically 100 - 400keV ) is
collimated by magnetic lenses and allowed to pass through a specimen under high vacuum.
The transmitted beam and a number of diffracted beams can form a resultant diffraction
pattern, which is imaged on a fluorescent screen kept below the specimen. The diffraction
pattern gives the information regarding lattice spacing and symmetry of the structure under
consideration. Alternatively, either the transmitted beam or one of the diffracted beams can
be made to form a magnified image of the sample on the viewing screen as bright-and dark-
field imaging modes respectively, which give information about the size and shape of the
micro-structural constituents of the material. High-resolution image, that contains
information about the atomic structure of the material, can be obtained by recombining the
transmitted beam and diffracted beams together.

Instrumentation: The instrumentation is very large and comprises of tungsten filament or

LaB6 or a field emission gun as source of electron beam, objective lens, imaging lens, CCD
camera, monitor, etc.

Applications: Transmission electron microscopy is used to study the local structures,

morphology, and dispersion of multicomponent polymers, cross sections & crystallization of
metallic alloys and of semiconductors, microstructure of composite materials, etc. The
instrument can be extended to include other detectors like Energy Dispersive Spectrometr
(EDS) or Energy Loss Spectrometer (ELS) to study about the local chemistry of the material
similar to SEM technique.

Disadvantages: The instrumentation is complicated and needs high vacuum. Sample

preparation is very time consuming. Some materials, especially polymers, are sensitive to
electron beam irradiation which results in the loss of crystallinity and/or mass.
 29

Fig: 18

Field Emission Gun

Collimators

Objective Lenses

Sample

Monitor

Imaging
Lens CCD Camera

Fluorescent
Screen

7.3 Scanning Probe Microscopy ( SPM ):

The scanning probe microscopy is a general term for a wide variety of microscopic
techniques, which measure the morphology and properties of surfaces on the atomic scale.
This includes the following:
Scanning Tunneling Microscopy (STM) – which studies the surface topography and electronic
structure;
Atomic Force Microscopy (AFM) – which studies the surface topography, surface hardness
and elastic modulus;
Lateral Force Microscopy (LFM) – which studies the relative frictional properties;
Scanning Thermal Microscopy (SThM) – which studies the thermal conductivity;
Magnetic Force & Electric Force Microscopy (MFM & EFM) – which study the magnetic
and electric properties;
The techniques of STM and AFM are discussed below, since these are widely used:

Principles: The general principle for all the scanning probe microscopes is that a sharper
probe (or a very fine tip) is used to scan the surface of the sample with much lower force and
obtain the topography and morphology information.
 30

Scanning tunneling microscope: When a sharp tip made of a conducting material is brought
close to a conducting sample, overlapping of the electron clouds between the two surfaces
will occur. If a potential is given between them a current of electrons is formed, which is
often referred as “tunneling” current, and the effect is known as “tunneling” effect. This
effect is largely depended on the distance between the tip and the sample material. Hence, if
the scanning tip is controlled by a high precision motion device made of piezo-electric
material, the distance between the tip and the sample can be measured during a scanning
through a feedback loop control of the piezo-electric element. By this way the sample can be
scanned by the tip with sub-angstrom precision.

Atomic force microscope: This technique operates by measuring the forces between the
sample and the tip, and the sample need not be a conducting material. Here, the tip is brought
close enough to the sample surface to detect the repulsive force between the atoms of the tip
material and the sample. The probe tip is mounted at the end of a cantilever of a low spring
constant and the tip-to-sample spacing is held fixed by maintaining a constant and very low
force on the cantilever. Hence, if the tip is brought close to the sample surface, the repulsive
force will induce a bending of the cantilever. This bending can be detected by a laser beam,
which is reflected off the back of the cantilever. Thus by monitoring the deflection of the
cantilever, the surface topography of the sample can be tracked. Since the force maintained
on the cantilever is in the range of inter-atomic forces (about 10-9 Newton), this technique
derived the name “atomic force” microscopy.
AFM operates at two modes:
Repulsive or contact mode – which detects the repulsive forces between the tip and sample;
Attractive or non-contact mode – which detects the van der waals forces that act between the
tip and sample.

Instrumentation:
Scanning tunneling microscope: It mainly consists of a scanner, probe motion sensor
composed of piezo-electric material, micro probe, etc.

Fig: 19

Piezo-electric
sensor Electronics Computer
Interface

Micro-probe

Sample

Atomic force microscope: It mainly consists of a scanner, cantilever, laser source, photo-
diode detector, micro-probe, etc.
 31

Fig: 20

Laser
Detector Electronics Computer
Interface

Sample

Cantilever Micro-probe

Applications: Both STM and AFM find applications widely in material sciences especially
for surface studies on a nano scale range. While STM finds its applications in the
characterization of surface structure (including the electronic structure), AFM finds its
applications in measuring the hardness of materials. Sometimes, AFM can be used in the
study of “depth profile” of the deposited oxide layer on to a material.

Disadvantages: A limitation to STM is that it can study only the conducting samples, since
the technique is based on the tunneling current between two conducting areas. Hence, it
doesn’t lend itself to the study of non-conducting materials. In fact the AFM had been
developed to encounter this problem. These methods require special sample preparation
techniques, which are tedious, like, thin sectioning, electo-polishing, various mechanical
cutting and polishing techniques, etc.

8. ANALYSIS THROUGH ELECTRO - CHEMICAL TECHNIQUE

Under this heading there are so many techniques which are used for analytical purposes like
Potentiometry, Amperometry, Conductometry, Electrogravimetry, Coulometry, etc.,. But,
here let us discuss few techniques that utilise modern instrumentation and find wide
applications. Some of the terms and definitions may be difficult to understand without prior
knowledge in electro-chemistry. So, in that case, students are advised to read appropriate
basic chemistry textbooks for having a clear idea.

8.1 POLAROGRAPHY
Principles: This technique involves the measurement of the current flowing in an electrolysis
cell due to the oxidation - reduction reactions (redox reactions) of the analyte substance
present in the solution. This redox reaction usually occurs at the surface of one of the
electrodes. The current that produced in this way is directly proportional to the concentration
of the components under study.
 32

Instrumentation: The system here to measure the current flow comprises of three electrodes
and differs from other normal electrochemical systems which normally contain only two
electrodes. This three-electrode system includes the following:

Working electrode - where the oxidation or reduction processes of the analyte of interest
occurs. It consists of liquid mercury flowing through a very narrow
bore capillary tube and is called “dropping mercury electrode” and
abbreviated as DME.

Reference electrode - this electrode is like silver-silver chloride (Ag/AgCl ) electrode and is
crucial for the precise control of the potential of the working electrode.

Auxillary electrode - the use of this electrode is to carry the bulk of the current and counters
the process that occurs at the working electrode and make it free from
any disturbances except to maintain the redox reaction. Hence, this
electrode is also called as “counter” electrode. Though this electrode is
not important analytically, it is important not to allow the products of
reaction that occur here to interfere with the process that occurs at the
working electrode. For this reason it is often placed in a separate
chamber with a fritted glass disc allowing electrical contact with the
rest of the cell, but not allowing diffusion of undesirable species to the
working electrode.

In the instrumentation of this technique these three electrodes are systematically combined to
an electrical integrator called “polarograph”. The modern polarography instruments have
several modifications as sampled dc polarography, pulse polarography and differential
polarography. For each method the mode of measurement of the current and its manipulation
varies while the basic concept is maintained. The following is the concept of polarography:
 33

Fig: 21

Mercury Reservoir

Reference Electrode Auxilliary

Electrode

Sample+ Fritted
Electrolyte Glass
Polarograph

Working Electrode

The sample is placed in a glass container with a medium, which consists of high concentrations of
electrolyte. This excess electrolyte helps in bringing down the potential of the electrode process to the
desired range and eliminates the interference caused by unwanted complexations and other reactions
within the system. This is also referred as “background electrolyte”.

Applications: It is widely used for the quantitative and qualitative determination of metals
and metal complexes as well as organic compounds in trace levels.

Disadvantages: The measurements with this technique are very sensitive to solution
composition, dissolved oxygen and capillary characteristics. Further, impurities if any,
present in the background electrolyte also affect the sensitivity.
 34

8.2 ELECTROPHORESIS
Principles: This technique is actually a separation process by applying an electric field. The
principle is that under the influence of an electric field dissolved ions present in a solution
will migrate at varied rates and direction in a column or a surface. At this instance two events
take place:
- ions of opposite charge will migrate in different directions and become separated on that
basis.
- ions of like charge , while migrating in the same direction, become separated due to
different migration rates.
Factors influencing migration rates are charge values and different mobility. Further, the
mobility of an ion is dependent on the size and shape of the ion, as well as the nature of the
medium through which it migrates. The medium used in most of the cases is either cellulose
or gel. This technique is sometimes referred as “Zone Electrophoresis”.

Instrumentation: The materials needed for the basic instrumentation are, cellulose or
polymeric gel as a supporting medium, enclosed tank with electrodes and buffer reservoirs
and dc power supply. The following is the illustration of the concept:

Fig: 22
Outer Cover Medium Porous Diffusion
Barrier

Buffer

_ +

Positive Ions Sample location Negative Ions

Applications:This technique finds application in the qualitative characterization of

biologically active materials, where, charged amino acids and other biomolecules need to be
separated. Thus, analysis of protein and nucleic acid using this method becomes very
popular.
This is highly useful for clinical and forensic work, where small amounts of complex samples
may be involved.

Disadvantages: This technique gives less precision results for quantitative work, hence,
application in this aspect is restricted. Mobility is very sensitive to supporting medium, so
selection of the medium is very important.
 35

9. ANALYSIS THROUGH OTHER TECHNIQUES

9.1 TOTAL ORGANIC CARBON ANALYZER (TOC)

The element carbon is the most common form that can be found everywhere. In certain
cases its measurement at trace levels is very important. Especially, in the fields of
Environmental Pollution, Pharmaceuticals, Industrial Effluents, etc., its measurement is very
vital to maintain the threshold limits of the concerned contaminants. Of these, monitoring the
organic compounds in the environmental studies (mostly which present in the water) is
important, since, inorganic carbon constitutes to only a lesser extent having its presence with
carbonates, bicarbonates and dissolved carbon dioxide. Hence, it is customary to find out the
presence of carbon as Total Organic Carbon, which represents the quantity of carbon present
in water as organic matter, either dissolved or suspended.

The principle of the TOC is very simple which involves complete oxidation of carbonaceous
materials to carbon dioxide and water by catalytic combustion or by chemical oxidation. The
released carbon dioxide is measured using a IR detector since this molecule strongly absorbs
in the IR region. In cases, wherein the measurement of inorganic carbon is necessitated then
it is done within the same instrument by purging air through the sample placed in acid
solution. This will create the formation of carbon dioxide and water from the inorganic
carbonates and bicarbonates. This is one of the simplest instruments in operation with the
chemical analysis.

9.2 ELEMENTAL ANALYZER

This technique determines the presence of the elements like Carbon, Hydrogen and
Nitrogen in a given substance and gives the result as percentage amount of these atoms
against the total weight. Since this technique specifically determines these three elements this
instrument is also called as “CHN Analyzer”. Most of the organic compounds are made up of
these three elements and oxygen, hence, after determining these three elements the
percentage weight of oxygen can be easily calculated.

This is a very important study when it comes to a newly synthesised compound to prove its
composition exactly. The principle of the instrumentation is that the substance under study is
combusted under oxygen stream in a furnace at high temperatures. The end product of the
combustion would be mostly the oxides of the concerned elements in the form of gases.
These are then separated and carried to the detector using inert gases like helium or argon.

It is one of the few analytical techniques that give a clear quantitative measurement of the
carbon, hydrogen and nitrogen. It finds applications in almost every field of chemistry like in
the analysis of organics, polymers, pharmaceuticals, energy (fuels), environmental studies,
etc.

9.3 POLARIMETRY
Principles:In the preceding section under spectroscopy (Section 3) we dealt with the
interaction of light with the substances leading to absorption, emission and fluorescence.
Here the phenomena concerns with the rotation of the plane of the plane polarised light when
 36

it is passed through the samples which lacks symmetry (e.g. sugar), these substances which
are asymmetric in nature are said to be optically active substances. A plane polarised light is
that which essentially has its vibration in only one direction or one plane as shown below:

Fig:23a

A B C

In the above illustration “A” refers to the light waves propagating in all directions or planes
(Multidirectional), “B” refers to the Nicol prism which cuts all the planes of light and allows
the light to come out with vibration in only one plane or direction (Unidirectional) and “C” is
the resultant light which is said to be a plane polarised light. It is the light, which is utilised
for the measurement of the optical activity of a compound. The plane of this light will be
rotated to certain extend depending on the nature of the compound under study and forms the
basis of this technique.

Instrumentation: The instrument setup is very simple and comprises of sodium lamp, nicol
polarizer, sample tube, nicol analyzer and an eyepiece. The set up is as follows:

Fig:23b

Sodium Lens Nicol Protective windows Sample Nicol Eyepiece

Lamp Polarizer Tube Analyzer Lenses

If, the rotation of the plane by a compound is in the clockwise then it is said to be dextro,
if it is in the anticlockwise then it is said to be laevo. For any compound the rotation depends
on the concentration and the length of the sample tube and importantly on the temperature
too.

Applications: It is mainly used as a quantitative tool. It finds extensive application in the

analysis of sugar in the industry. In pharmaceutical industry it is used for the measurement of
concentration of optically active drugs.
 37

Disadvantages: It requires the samples only in solution form. The sample tube, after filling
with the sample solution, should be free of bubble or any free particle, otherwise the light
path will be affected and hence the accuracy. The instrument should be thermostatted, since
the optical activities of the substances vary with temperature.

9.4 UV/VISIBLE SPECTROPOLARIMETRY

Principles: In Section 9.3, the rotation of the plane of the polarized light by optically active
compounds has been discussed. In practice, it is produced by combining two plane-polarized
light waves of identical frequency moving through the same region of the space in the same
direction, to produce a resultant wave, which is also linearly polarized!. If these two waves of
identical amplitude are orthogonal, combining them can lead to linearly polarized or
circularly polarized light depending on the phase difference between the two waves.

Optical Rotatory Dispersion (ORD):

In polarimetry, the rotation of the plane of linearly polarized light (two combined plane-
polarized waves with no phase difference) by optically active substances is studied at a fixed
wavelength. In spectropolarimetry it is studied over a range of wavelength whereby the
dependence of the rotation on the selected wavelength range is measured and is termed as
Optical Rotatory Dispersion. The instrumentation is similar to a polarimeter except for a
tunable wavelength source. ORD (and polarimetry) measures the differences in refraction of
the polarized light waves through the sample resulting in a rotation of the plane.

Circular Dichroism (CD): If the two plane-polarized orthogonal light waves are 900 out of
phase then the resultant gives a circularly polarized light. It can either be right circularly
polarized (d-component) or left circularly polarized (l-component) depending on the
direction of the phase shifting. Again, if these two oppositely polarized circular waves are
combined the resultant would be a linearly plane-polarized light! CD measures the
differences in absorbance of the polarized light waves by the sample resulting in an
ellipticity, when the two circularly polarized light waves are combined after passing through
the sample.

Linear Dichroism (LD): The linearly polarized light can either be made parallel or
perpendicular to the orientation axis using a polarizer and, if this is passed through the
sample it will give the information regarding the orientation of the molecules present in the
sample. LD measures the differences in absorbance of the linearly polarized light waves.

Instrumentation: Normally the instrumentation of a spectropolarimeter is so designed to

accommodate both the functions of ORD and CD. It consists of a source (covering UV to
visible), monochromator, polarizer, analyzer, detectors, lock-in amplifier, display device, etc.
 38

Fig: 24

ORD
Detector
Source Monochromator Polarizer Sample Analyzer

CD/LD
Lock-In Amplifier Detector

Analog Ratio Display

Device

Applications: Both CD and ORD measurements are employed in fundamental studies to

provide structural information in the area of stereochemistry and conformation about
optically active compounds like, amino acids, proteins, etc. LD measurement is mostly
employed to study the molecular orientations in the stretched polymer films.

Disadvantages: Only optically active compounds can be analyzed and requires the samples in
liquid form.

9.5 VIBRATIONAL CIRCULAR/LINEAR DICHROISM (VCD/VLD)

Principles: The phenomenon of CD and LD if occurs in the Infra-Red region which

corresponds to the vibrational frequencies of the molecules then the techniques are termed as
Vibrational Circular Dichroism (VCD) and Vibrational Linear Dichroism (VLD).

Instrumentation: VCD spectrometer is constructed using the same detection scheme as

described for the UV/Vis CD spectrometer described above, except for the optical
components suitable for infrared region. Thus normally VCD accessory is combined with a
mid-IR FTIR spectrometer.

Fig: 25
Source Monochromator Polarizer Analyzer CD/LD
Sample
Detector

Lock-In Amplifier

Analog Ratio
Display Device
 39

9.6 MASS SPECTROMETRY

Principle: In this technique chemical substances are bombarded with high energy electron
beam causing total destruction and fragmentation of the molecules in the sample. This
fragmentation results in small charged “pieces” or fragments of the molecules, which,
because of their charge are made to move through a magnetic field. The magnetic field
affects each fragments differently based on their mass and charge that they carry and thus
they are separated.

Instrumentation: Source of high-energy electrons, ion accelerator, magnets, detector,

recorder and high vacuum pumping system. In the instrumentation the entire path of the
fragments, including the inlet system must be evacuated. The reason is to avoid collisions of
both the electron beam and the sample ions with contaminating foreign particles which would
otherwise affect the sensitivity and hence the results. For this technique there are two
different instrument designs as Magnetic Sector Mass Spectrometer and Quadrupole Mass
Spectrometer. The former is the older version which uses strong magnets to focus the
fragmented ions to the detector plate, whereas, the latter uses four short parallel metal rods,
two vertical rods are connected to the positive pole and the other two to the negative pole of a
variable power source. This will create a variable electric field and is used to focus the
fragments on to the detector slit. This quadrupole instrument is newer and more popular
because of its compact design and also it provides a faster scanning capability. The following
is the illustration for the quadrupole mass spectrometer:

Fig: 26

Electron Beam Slit

Sample
Introduced

Fragment Path Metal Rods Ion Detector

Applications: It is widely used in conjunction with IR, UV and NMR in the identification and
structural analysis of organic compounds. It is also possible to determine the trace impurities
in a wide range of inorganic materials. It is well suited for gas analysis and is used by the
petroleum industry for both the qualitative and quantitative analysis of hydrocarbon
distillates and other petrochemicals. It is invaluable in the analysis of both terrestrial and
extra-terrestrial atmospheres, the latter being achieved by instruments with light-weight
 40

components. The mass spectrometer has been used as a detector in gas chromatography ( GC-
MS ), in liquid chromatography ( HPLC-MS), in thermogravimetry ( TGA-MS ) and in
inductively coupled plasma spectrometry ( ICP-MS ) – which is normally called as
“hyphenated” technique.

Disadvantages: The instrumentation is complex and difficult to maintain.

9.7 LASER LIGHT SCATTERING SYSTEM

When light (usually a laser beam) is shined or focused on a solution containing the
macromolecule of interest (polymers, proteins, etc.,), it will scatter and the scattered light
give us information about molecular structure and motion in the solution.

Principles: Interaction of light (electro-magnetic radiation) with matter results in two

processes, viz. absorption (which forms the basis of spectroscopy) and scattering. When a
beam of light falls upon a matter, the electric field associated with the light polarizes the
electron cloud of the atoms. Thus a dipole is induced in the molecules, which oscillates with
the electric field. These oscillating electron clouds then serve as secondary sources of light
and emit light in various directions (scattering). The scattered light has almost the same wave
length as the incident light (also referred as Elastic Scattering or Rayleigh Scattering) and
merely redirected from the incident beam. It is the intensity of the scattered light and its
angular distribution which give the information regarding the nature of the molecules under
study.

Instrumentation: It consists of a laser source, thermostatted sample holder, movable photo

diode detector, correlator, etc.,.

Fig: 27

Laser Beam Sample Holder

Laser Source

Moveable
Detector
 41

Applications: Usually the instrument operates at two modes viz. Static Light Scattering
mode and Dynamic Light Scattering mode.
Static Light Scattering – in this mode, instrument measures the intensity and angular
dependence of the scattered light ( ie. Elastic or Rayleigh Scattering ). This gives the
information on molecular mass, radius of gyration and second virial coefficient ( interaction
of solvents with the substances under study ). Thus it is helpful in characterising
macromolecules and their associations.
Dyamic Light Scattering – in this mode instrument measures the fluctuations in the intensity
of the scattered light caused by the continuous motion of the particle ( Brownian motion ).
Continuous movement of particles in the medium will cause the frequency of the scattering
light to change ( Doppler shift ) and this type of scattering is sometimes referred as Quasi-
Elastic Light Scattering ( QELS ) and the technique Photon Correlation Spectroscopy. The
measurement gives the information on the translational diffusion coefficient and the
polydispersity of the sample, which are useful in determining the macromolecule size and
their aggregations.
 42

10. SUMMARY
The following is the summary of the foregone descriptions:

Name of the Technique General Applications

SPECTROSCOPY
Atomic Absorption and To analyse alkali and alkaline earth metals in dilute
Emission Spectrometry solution, natural liquids, and extracts at trace levels
UV-Visible Spectrometry To analyse molecular (organic) and ionic species capable
of absorbing at UV or Visible wavelengths in dilute
solutions
Fourier Transform Infra-Red To analyse only molecular compounds (organic
Spectrometry (FTIR) compounds, natural products, polymers, etc.)
Raman Spectrometry To analyse molecular (organic) compounds which are not
responding well in the IR region and hence, it is an
alternate to IR
Nuclear Magnetic Resonance To identify and characterize the organic and inorganic
Spectrometry compounds
Microwave Spectrometry To analyse simple gaseous molecules in Far IR region,
to study their stereo chemistry
Electron Spin Resonance To study the formation and life time of the free radicals
Spectrometry formed in organic reactions and also finds applications in
biological works
Fluorometry (Molecular To study the molecular and ionic compounds in dilute
Flourescence) solutions capable of giving flourescence, finds
applications in vitamin analyses

CHROMATOGRAPHY
High-Performance Liquid To separate and analyse complex mixtures or solutions
Chromatography which include liquids and solids of both organic and
inorganic origins
Gas Chromatography To separate and analyse mixtures of volatile organic
compounds, solvent extracts and gases

THERMAL ANALYSIS
Thermogravimetric Analysis To study the mass changes of materials like polymers,
glasses, ceramics, etc., such as evaporation,
decomposition, gas absorption, de-sorption, dehydration,
etc.
Thermomechanical Analysis To study the glass transition temperature and the
expansion coefficient of composite and laminate
materials
Differential Thermal Analysis To study the exothermic and endothermic behaviour of
clay materials, ceramics, ores, etc.
Differential Scanning To study the curing process of the thermoset polymers
Caloriemetry and heat of melting of thermoplastic polymers.
 43

X-RAY TECHNIQUES
X-Ray Fluorescence To identify the elements and their valence states present
Spectrometry and X-Ray Photo- in the surface of the materials
emission Spectrometry
X-Ray Diffractometry To study the crystalline properties of solid substances

MICROSCOPY
Scanning Electron Microscopy To study the topography, electronic structure and
compositions of metals, ceramics, polymers, composites
and biological materials
Transmission Electron To study the local structures, morphology, and dispersion
Microscopy of multicomponent polymers, cross sections and
crystallizations of metallic alloys, semiconductors,
microstructure of composites, etc.
Scanning Probe Microscopy To characterise and to study the hardness of materials
like ceramics, polymers, composites, etc., on a nano-
scale range

ELECTRO-CHEMICAL TECHNIQUES
Polarography To study and determine metals, metal complexes and
organic compounds in trace levels
Electrophoresis To study and characterize biologically active compounds
like proteins, amino acids and other bio-molecules

OTHER TECHNIQUES
Total Organic Carbon Analyzer To monitor pollutants in environmental studies by
determining the carbon contents of the trace compounds
Elemental Analyzer To estimate percentage compositions of elements like
carbon, hydrogen and nitrogen present in newly
synthesised organic compounds
Polarimetry To analyse and quantitate optically active compounds
like
sugar
UV/Visible Spectropolarimetry To get the structural information of optically active
(CD//ORD) compounds like, amino acids, proteins, etc.
Vibrational Circular Dichroism Same as above but in the IR region. VLD measurement is
(VCD/VLD) employed to the molecular orientations of thin polymer
films
Mass Spectrometry To identify the organic compounds. Often used as
detectors with HPLC and GC
Laser Light Scattering System In the study of macromolecules like polymers, gels,
proteins for determining molecular mass & size and their
associations
 44

11. REFERENCE

1. G. W. Ewing, Instrumental Methods of Chemical Analysis, Fourth Edition,

McGraw-Hill, New York, 1975.

2. J. Kenkel, Analytical Chemistry - Refresher Manual, Lewis Publishers, Boca Raton,

1992.

3. J. Kenkel, Analytical Chemistry for Technicians, Second Edition, Lewis Publishers,

Boca Raton, 1994.

4. F.W. Fifield and D. Kealey, Principles and Practice of Analytical Chemistry, Second
Edition, International Textbook Company Limited, London, 1983.

5. D. A. Skoog, D. M. West and F. J. Holler, Fundamentals of Analytical Chemistry,

Sixth Edition, Saunders College Publishing, Philadelphia, 1992.

6. J. P. Sibilia, Materials Characterization and Chemical Analysis, Second Edition,

VCH Publishers, New York, 1996.

7. B. G. Yacobi, D. B. Holt and L. L. Kazmerski, Microanalysis of Solids, Plenum Press,

New York, 1994.

8. J. W. Robinson, Undergraduate Instrumental Analysis, Fifth Edition, Marcel Dekker,

New York, 1995.

9. T. Kuwana, Physical Methods in Modern Chemical Analysis, Volume 2, Academic

Press, London, 1980.

10. A. Weissberger, Physical Methods of Organic Chemistry, Volume I – Part 3,

Interscience Publishers, New York, 1960.

11. J. F. Rabek, Experimental Methods in Polymer Chemistry, John Wiley and Sons Ltd,
Chichester, 1980.

12. E. Schroder, G. Muller and K. F. Arndt, Polymer Characterization, Hanser Publishers,

Munich, 1988.

13. H. G. Brittain, Chiroptical Techniques in Analytical Instrumentation Handbook, Editor:

G. W. Ewing, Marcel Dekker, New York, 1990.

14. J. D. Ingle and S. R. Crouch, Spectrochemical Analysis, Prentice-Hall Int., New Jersey,
1988.

15. A. Rodger and B. Norden, Circular Dichroism and Linear Dichroism, Oxford University
Press, Oxford, 1997.