UNIT-1

ELECTROMAGNETIC RADIATION (EMR):

 Initially electricity and magnetism were considered to be separate forces. Later in
the year 1873, James Clerk Maxwell, the Scottish physicist, developed a combined
theory known as electromagnetism. An electromagnetic field produces
electromagnetic radiation also referred to as EM radiation.
 Electromagnetic radiation is a form of energy that is present all around us and takes
various forms like microwaves, television waves, radio waves, gamma rays, X-rays,
etc. In this piece of article, we will discuss electromagnetic radiation and its
properties.
 EMR is a wave that propagates (radiates) through a vacuum at the speed of light
(just under 300 000 m/s) and transfers energy from one place to another.
 These waves carry energy as synchronized oscillations of electric and magnetic fields
that are perpendicular to each other and perpendicular to the direction of travel
 Although it is a wave, it also can be detected as discrete ‘particles’ of light called
photons.
 Electromagnetic radiation can be defined as a form of energy that is produced by
the movement of electrically charged particles travelling through a matter or vacuum
or by oscillating magnetic and electric disturbances.
 The magnetic and the electric fields come at 90° to each other, and the combined
waves move perpendicular to both electric and magnetic oscillating fields occurring
during the disturbance.

ELECTROMAGNETIC THEORY
Magnetism and electricity were once considered as separate forces. However, in the year
1873, Clerk Maxwell, a Scottish physicist developed a unified theory of electromagnetism.
Its study deals with how the electrically charged particles interact among themselves and
with the magnetic field. The main electromagnetic interactions are provided in the points
mentioned below.
 Magnetic poles come in pairs that repel and attract each other, just like electric
charges do.
 The force of repulsion or attraction between two electric charges is inversely
proportional to the square of the distance between the particles.
 An electric field in motion produces a magnetic field.
 A wire with an electric current produces a magnetic field whose direction depends
on the direction of the electric current.
PROPERTIES OF ELECTROMAGNETIC RADIATION
 When electromagnetic radiation occurs, it is released as photons. These are bundles
of light energy or quantized harmonic waves which travel at the speed of light. Then
based on the wavelength of the electromagnetic spectrum, the energy is grouped
into different categories.
 These magnetic and electric waves travel perpendicular to each other and have some
characteristics like wavelength, amplitude, and frequency. Some basic properties of
Electromagnetic Radiation are given in the points mentioned below.
 They can travel through empty space. Waves other than electromagnetic waves have
to travel through some substance. For example, sound waves will need either a solid,
liquid or gas to pass through.
 The speed of light which is 2.99792458 x 108 m/s is always constant.
 Wavelength is commonly characterized by the symbol ‘λ’. It is defined as the distance
between two most near points in phase with each other. Hence, two adjacent crests
or troughs on a wave are separated by a distance of a single complete wavelength.
 EMR radiated by atomic particles at the source (Sun)
 Propagates through the vacuum of space at the speed of light
 Interacts with the Earth’s atmosphere
 Interacts with the Earth’s surface
 Various optical systems and detectors
Electromagnetic radiation or energy can be described through three properties:

 Wavelength —
 Frequency —

ELECTROMAGNETIC SPECTRUM

 Electromagnetic spectrum is the range of frequencies EM radiations along with their

associated wavelengths and photon energies. It consists of Gamma-rays, X-rays,
ultraviolet rays, infrared rays, radio waves and microwaves.
 Electromagnetic radiations have a wide range of frequencies, wavelengths and
photon energy levels. These waves travel at the speed of light in vacuum.
WAVES AND THEIR CHARACTERISTICS/PARAMETERS:
 Electromagnetic radiation occurs when an atomic particle, like an electron, is
accelerated by an electric field, causing it to accelerate. Electromagnetic waves and
their characteristics is explained briefly in the points mentioned below.
WAVELENGTH: Wavelength (λ) is the distance between successive crests of a wave,
especially points in an electromagnetic wave or sound wave. It can be simply defined as
the distance of one full cycle of the oscillation. If ‘λ’ is the wavelength, ‘c’ is the speed of
light and ‘ν‘ is frequency. Then we can derive the relation given below.
c=λν
Describes the shape and movement of the wave and is a measure of the distance between
repetitions of the shapes of the wave such as valleys, peaks and zero-crossings. This is
one way of perceiving the wave through instruments and other sensors. For example, the
visual characteristics of visible light such as color and visibility is dictated by the
wavelength. The smallest wavelengths have been measured to be smaller than the size of
an atom, while the largest are bigger than the diameter of our planet.
The shorter the wavelength, the greater the frequency and the greater the frequency, the
higher the energy.
Wavelength units: length Angstrom (A) : 1 A = 1x10-10 m; Nanometer (nm): 1 nm=1x10-9
m; Micrometer (µm): 1 µm = 1x10-6 m;
AMPLITUDE: It is the distance from the middle of the wave to the maximum vertical
displacement of the wave. Larger the amplitude, the higher the energy and the lower the
amplitude, the lower the energy. Amplitude tells us about the brightness or intensity of a
wave compared to other waves. Amplitude is the distance from the maximum vertical
displacement of the wave to the middle of the wave. This measures the magnitude of
oscillation of a particular wave. In short, the amplitude is basically the height of the wave.
Larger amplitude means higher energy and lower amplitude means lower energy.
Amplitude is important because it tells you the intensity or brightness of a wave in
comparison with other waves.
FREQUENCY: Describes the number of crests and falls or peaks and valleys that pass
through a point in one second. The measurement unit for one cycle per second is the
Hertz, after the man that established the existence of radio waves, Heinrich Hertz.
The number of cycles per second is defined as Frequency. It is defined as Hertz (Hz) or sec -
1. If ‘E’ is the energy, ‘h’ is Planck’s constant which is equal to 6.62607 x 10 -34 and ‘ν‘ is

the frequency we can derive the relation given below.

E = hν
Thus, we can see that frequency is directly proportional to the energy.
PERIOD: Period is commonly characterised by the symbol ‘T’. It is the total time that a
wave takes to travel 1 wavelength.
VELOCITY: In relation to electromagnetic radiation, the velocity is normally expressed as:
Velocity = λν
[where, ν = frequency]
The wave velocity in vacuum for electromagnetic wave is = 186,282 miles/second or
2.99×108 m/s.

WAVENUMBER: it is defined as a count of the number of wave crests (or troughs) in a

given unit of length (symbolized by ν): Wavenumber units: inverse length (often in cm-1)
ν = ν ~ /c = 1/λ
FREQUENCY UNITS: Unit cycles per second 1/s (or s-1) is called hertz (abbreviated Hz).
Unit Frequency, (cycles/sec) Hertz, Hz 1 Kilohertz, KHz 103 Megahertz, MHz 106
Gigahertz, GHz 109.
ENERGY — Describes the intensity of the EMR through electron volts, which is commonly
used for energetic or active EMR, like gamma rays and X-rays.
 ELECTROMAGNETIC RADIATION
 Electromagnetic Radiation (EMR) or Electromagnetic energy is the energy
propagated in the form of an advancing interaction between electric and magnetic
fields. It travels with the velocity of light. Visible light, ultraviolet, and infrared rays,
heat, radio waves, X-rays all are different forms of electromagnetic energy.
 EMR ranges from gamma rays with very short wavelength to long radio waves. The
shortest wavelengths can also be modelled as particles (photons). The interaction of
EMR with matter forms the basis for Remote Sensing.
 Electric and Magnetic fields that are perpendicular to each other and
propagation to the direction of travel. Although it is a wave, it also can be detected
as discrete particles of light, called photons.
 Electromagnetic radiation (EMR) is a form of radiated or transported energy that
does not require a medium in order to propagate, unlike mechanical waves such as
sound and vibrations. Mechanical waves travel by transferring energy through
molecular contact, causing molecules to bump into each other in order to transfer
kinetic energy which can be observed visually in water ripples.
 Electromagnetic waves are created by magnetic and electric fields coupling together
to form waves, usually released by certain electromagnetic processes. The most
common examples of electromagnetic radiation are visible light and X-rays.
 Electromagnetic radiation is also known as electromagnetic waves.
 RADIO WAVES: Radio waves primarily come from the oscillation of currents in
conductors. The long wavelengths travel easily and are not interfered by atoms
(Except conductors). Radio Waves are approximately 103 m in wavelength. As the
name implies, radio waves are transmitted by radio broadcasts, TV broadcasts, and
even cell phones. Radio waves have the lowest energy levels. Radio waves are used
in remote sensing, where hydrogen gas in space releases radio energy with a low
frequency and is collected as radio waves. They are also used in radar systems,
where they release radio energy and collect the bounced energy back. Especially
useful in weather, radar systems are used to can illustrate maps of the surface of
the Earth and predict weather patterns since radio energy easily breaks through the
atmosphere.
 MICROWAVES: The Microwaves are very short wavelength compare to radio waves.
Microwaves are associated with the vibrations of some atoms and molecules. Water
molecules act as a dipole. Microwaves can be used to broadcast information through
space, as well as warm food. They are also used in remote sensing in which
microwaves are released and bounced back to collect information on their
reflections. Microwaves can be measured in centimetres. They are good for
transmitting information because the energy can go through substances such as
clouds and light rain. Short microwaves are sometimes used in Doppler radars to
predict weather forecasts.
 INFRARED WAVES: Wavelengths longer than visible light are called Infrared rays.
Molecules are heated by Infrared waves. Infrared radiation can be released as heat
or thermal energy. It can also be bounced back, which is called near infrared
because of its similarities with visible light energy. Infrared Radiation is most
commonly used in remote sensing as infrared sensors collect thermal energy,
providing us with weather conditions.
 VISIBLE LIGHT: Visible Light is radiation in a narrow band of wavelength. Many
atoms and molecules have specific behaviour at unique frequencies of light.
Corresponds to maximum output from the Sun. Visible Light is the only part of the
electromagnetic spectrum that humans can see with an unaided eye. This part of
the spectrum includes a range of different colors that all represent a particular
wavelength. Rainbows are formed in this way; light passes through matter in which
it is absorbed or reflected based on its wavelength. Thus, some colors are reflected
more than other, leading to the creation of a rainbow.
 ULTRAVIOLET WAVES: Ultraviolet rays have shorter wavelengths than visible
light. Some molecules absorb ultraviolet and reemit visible light.
 X-RAYS: X-rays are associated with energetic transitions in atoms. The short
wavelength x-rays can penetrate materials. Electromagnetic radiation composed of
photons that carry minimum-ionization energy, or more, (which includes the entire
spectrum with shorter wavelengths), is therefore termed ionizing radiation. (Many
other kinds of ionizing radiation are made of non-EM particles). Electromagnetic-
type ionizing radiation extends from the extreme ultraviolet to all higher frequencies
and shorter wavelengths, which means that all X-rays and gamma rays qualify.
These are capable of the most severe types of molecular damage, which can happen
in biology to any type of biomolecule, including mutation and cancer, and often at
great depths below the skin, since the higher end of the X-ray spectrum, and all of
the gamma ray spectrum, penetrate matter.
 GAMMA RAYS: Gamma rays are photons associated with nuclear or particle
processes. Acceleration of a very energetic charged particle gives x-rays and gamma
rays, it’s Called bremsstrahlung. A gamma ray, also known as gamma
radiation (symbol γ or ), is a penetrating form of electromagnetic radiation arising
from the radioactive decay of atomic nuclei. It consists of the shortest wavelength
electromagnetic waves, typically shorter than those of X-rays.
With frequencies above 30 exahertz (3×1019 Hz), it imparts the highest photon
energy.
ELECTRONIC SPECTRA AND MOLECULAR STRUCTURE
 Molecules exhibit electronic spectra from transitions between electron energy levels.
These spectra are more complex than those of atomic spectra which involve
transitions between electron energy levels which typically produce sharp line
spectra.
 The energies associated with molecular electronic spectra (typically in
the optical or uv region) are typically much larger than those associated
with vibrational spectra (typically in the infrared) and rotational spectra (typically in
the microwave region).

 This contributes to the complexity of the electronic spectra since the transitions
from a multitude of vibrational and rotational levels produce many spectral lines, a
"band" of frequencies.
 Electronic transitions are essentially instantaneous, so there is no time for
appreciable motion of the nuclei.
 So the transitions appear as vertical lines with no change in internuclear distance.
This is referred to as the Franck-Condon principle.
 The spectra are strongly affected by the probability that an electron is at a location
to contribute to such a "vertical" transition.
 That probability is the wave function squared, and at least the lowest vibrational
states are approximated by the quantum harmonic oscillator.
 INTERNAL STANDARDS AND STANDARD ADDITION CALIBRATION METHODS
STANDARD ADDITION PROCEDURE:
1. Add one or more increments of a standard solution to sample aliquots of the same
size. Each mixture is then diluted to the same volume.
2. Prepare a plot of Analytical Signal versus:
a) volume of standard solution added,
b) Concentration of analyte added.
 External standard method is used when your unknown solution is prepared by
dissolving an amount of the analyte in pure solvent(s), but internal standard is used
when interference on the signal measured occurred due to matrix composition.
 A standard addition is a known quantity of analyte added to unknown to increase
the concentration of analyte.
INTERNAL STANDARD: It is a known amount of a compound different from analyte, that
is added to the unknown.
 Is a chemical substance that is added in a constant amount to samples and
calibration standards in a chemical analysis.
 Internal standard should be very similar, but not identical to the chemical
species of interest in the sample. It should be stable, must be inert, should have
same sample condition.
 An internal standard is a substance that is added in a constant amount to all
samples, blanks, and calibration standards in an analysis.
 Internal standard calibration involves the comparison of the instrument
responses from the target compounds in the sample to the responses of reference
standards added to the sample or sample extract before injection.
 The method of internal standards is used to improve the precision of quantitative
analysis. An internal standard is a known concentration of a substance that is
present in every sample that is analyzed. Internal standards can be used with
either the calibration curve or standard addition methods, although the former
is probably more common.
 The purpose of the internal standard is to behave similarly to the analyte but to
provide a signal that can be distinguished from that of the analyte. Ideally, any
factor that affects the analyte signal will also affect the signal of the internal
standard to the same degree. Thus, the ratio of the two signals will exhibit less
variability than the analyte signal.
 Internal standards are often used in chromatography, mass spectroscopy and
atomic emission spectroscopy. They can also be used to correct for variability
due to analyte loss in sample storage and treatment.
Example:
 In the analysis of sodium metals by flame atomic emission spectroscopy, lithium
may be used as an internal standard. Using the data below, calculate the
concentration of sodium in the sample. Compare the precision of the result from the
internal standard method with that achieved with the calibration curve method (ie,
if the lithium emission signals were ignored).
 UV-VISIBLE SPECTROSCOPY
The wavelength range of UV radiation is 200 nm – 400 nm. There are mainly two types of
UV region.
1. 190 nm to 400 nm that is called near ultraviolet region.
2. 400 nm to 800 nm that is called visible region.
3. Below 200 nm that is called far ultraviolet region.
 INSTRUMENTATION UV VISIBLE SPECTROSCOPY
SPECTROSCOPY:
 It is the branch of science that deals with the study of interaction of matter with
light.
 It is the branch of science that deals with the study of interaction of electromagnetic
radiation with matter.
 UV-Visible Spectrophotometer is an instrument design to measure the spectrum of
a compound.
PRINCIPLE OF UV-VISIBLE SPECTROSCOPY
 The principle is based on the measurement of spectrum of a sample containing
atoms/molecules.
 The UV radiation region extends from 190 nm to 400 nm and UV visible radiation
region extends from 400 nm to 800 nm.
 Absorption laws When a beam of monochromatic light passed through a solution
it may transmitted as a such or some of may be absorb.
The Qualitative & Quantitative determination of compounds by spectrometric technique
is based on two law.
1. Lamberts law
2. Beers law
Lamberts law: Its state that light absorbed by solution is directly proportional to length
of the light through the solution.
A=log(Io/I)= Є.l
where as,
A= absorbance
Є=molar absorptivity coefficient
l= path length of the sample (usually 1cm)
Beers law: its state that the amount of light absorbed is directly proportional to
concentration of absorbing solute in the solution.
A=log(Io/I)= Є.C

Combined Beer lamber Law is A=log(Io/I)= Є.C.l

A= absorbance
Є=molar absorptivity coefficient
l= path length of the sample (usually 1cm)
C=concentration of solution moles/liter
Io=Incident light
It=Transmitted light
Instrumentation Spectrophotometer have the following basic components.
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1. Source: A continuous source of radiant energy covering the region of spectrum in
which the instrument is designed to work.
Characteristics of an ideal source:
A. Gives constant intensity
B. Have low noise
C. Prolonged stability.
 UV- Hydrogen Deuterium lamp (190-420) nm, Life time approx. 2000hrs.
 Visible- Tungsten lamp (350-3000) nm, Lifetime approx. 10000hrs.
 Radiation source For UV H2 discharge lamp and deuterium lamp are used.
Deuterium lamp provide high intensity emission and adequate continuity in the
region of 190-380nm. For visible light tungsten filament lamp is used, Xenon
discharge lamp and mercury arch lamp. Tungsten filament provides a higher output
in a cross over region of 320-380nm and longer range up to 900nm.
2. Filters: Filters are two types
1. Absorption filter
2. Cut off filters
1. Absorption filters:
 They are made up of colored glass or dye.
 Transmit only a specific band of wavelength.
 They are inexpensive.
 Transmission is only about (10-20)%.
2. Cut off Filters:
 Transmission is about 100%
 Used to achieve only a specific band of wavelength.
 They are used in combination with absorption filters
 To decrease the bandwidth of absorption filters.
3. Monochromator: Monochromator It allows passing of radiation of particular
wavelength. It consists of two measures parts
Function: To produce a beam of monochromator (Single wavelength) radiation
that can be selected from a wide range of wavelengths.
Components:
1. Entrance Slit
2. Collimating device (to produce parallel light)
3. Dispersing device (grating, prism)
4. A focusing lens or mirror
5. An exit slit

4. Sample Holder/Containers: Sample containers are generally made up of plastic,

quarts and glass.
1. Plastic cuvette- used for fast spectroscopic assays, where speed is more
important than high accuracy.
 2. Glass cuvette- used in the wavelength range of visible light. - transmission range
340-2000nm
3. Quartz cuvettes- They are of two types: UV quartz- most common material. -
transmission range 190-2500 nm. IR quartz- rarely used - transmission range
190-3500 nm.
 Smallest capacity holding- 70 microliter.
 Largest capacity holding- 2.5ml or larger Sample Container
5. Detectors: A detector converts a light signal into an electrical [Link] should give a
linear response over a wide range with low noise and high sensitivity. Spectrophotometers
most commonly used photomultiplier tube detector. Light may or may not be absorbed by
passage through the sample. This is determined by quantitative measurement of light
passing through the instrument by means of detector such as photo multiplier tube, photo
voltaic cell, photo conductive cell and barrier layer cell.
1. Photomultiplier tube:
 Photo multiplier tube Photo multiplier tube is generally used in spectroscopy. It
consists of an evacuated tube containing one photocathode and 9 to 16 electrodes
known as dynodes.
 Photocathode is negatively charged and then a photon hits the photocathode.
Emission of electrodes takes place due to photovoltaic effect. When incident
radiation falls on the metal surface of cathode and get emitted which are attracted
towards the first dynode.
 These with are attracted by the second dynode and with are emitted by second
dynode. Hence, the process is repeated at all the dynodes. At the end of dynode
chain and anode is present which acts as collector of electrons.
 The current flowing from anode is directly proportional to photo electron flux
generated by photo cathode. Ejected photoelectron strikes dynode, secondary
electrons released, voltage accelerates secondary electrons to next dynode and so
on, then big voltage divider. Result is large charge packet hitting anode.
 2. Photovoltaic cell:
 It consists of semi-conductor like selenium which is deposited on a strong metal
base such as iron. A thin layer of gold or silver is sprayed over the semiconductor
which then acts as second collective electrodes. When radiations fall on the
surface, it produces electrons at the selenium silver interface. Since, barriers
exist between selenium and iron. The flow of electron is prevented which results
in accumulation of electron on the silver surface and produces electrical voltage.
 The Photovoltaic cell is the semiconductor device that converts the light into
electrical energy. The voltage induces by the PV cell depends on the intensity of
light incident on it. The name Photovoltaic is because of their voltage producing
capability.
 The electrons of the semiconductor material are joined together by the covalent
bond. The electromagnetic radiations are made of small energy particles called
photons. When the photons are incident on the semiconductor material, then the
electrons become energised and starts emitting.
 The energises electron is known as the Photoelectrons. And the phenomenon of
emission of electrons is known as the photoelectric effect. The working of the
Photovoltaic cell depends on the photoelectric effect.

TYPES OF INSTRUMENTS
1. Single Beam Spectrophotometer
2. Double Beam Spectrophotometer
FORENSIC APPLICATIONS OF UV VISIBLE SPECTROPHOTOMETER
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[Link] bags and plastic films
[Link] Drugs
[Link] analysis
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[Link] of impurities
[Link] fuels
[Link] and poison
[Link] solvents
[Link] Poisons
 Analysis of dyes and pigments from hair and fibers may appear identical in color
with human eye, the fibers may have been dyed with different pigments.
 Analysis of dyes form pen. A UV visible provides a mean a discriminating a
similarly colored inks.
 Analysis of drugs- various drug molecule produce characteristics UV-visible
spectra.
 Analysis of explosives- the organic explosives like nitro aromatic and nitro
amines show typical absorption in UV visible region.
 Analysis of paint-dyes and pigments.

IR SPECTROSCOPY
 Infrared spectroscopy (IR spectroscopy) is an important technique that deals with
the interaction of a molecule with IR range of the electromagnetic spectrum ranging
from 4000 to 400 cm−1.
 The necessary condition for a molecule or sample to show infrared spectrum is the
change in the electric dipole moment of the functional group present in a molecule
or a sample during the vibration based on the selection rule for IR transitions
 Molecules whose dipole moments change during vibration are infrared-active. IR
spectroscopy primarily helps in the identification of the type of chemical bonds
present in a sample that are reflected by the absorption of characteristic wavelength
of the infrared radiation responsible for the vibrational transition induced from
particular functional groups.
 The great advantage of infrared spectroscopy is that samples in the form of liquid,
gas, solid, powder, or film can all be studied with a careful selection of sampling
technique. The more advanced technique Fourier transform infrared (FTIR)
spectroscopy offers a facile kinetic and mechanistic insight of the chemical
functionalities responsible for any chemical change or a noncovalent
supramolecular interaction.
 These vibrational frequencies can be analyzed easily and quickly through high-
resolution spectra for both solid and liquid samples, and that can be done non-
destructively. The variations in the shape of characteristic peaks/bands along with
their intensity are crucial in predicting the chemical environment of the
functionalities present in the vicinity and dynamic changes occurring therein, which
unveils the kind of functionalities or ligand with which the samples are interacting.
Principle of IR Spectroscopy
 When IR radiation interact with sample molecule, the sample molecule will absorb
the specific applied IR light which is matched with the internal vibrational frequency
of the molecule and other residual frequencies are transmitted via the sample. Due
to absorption of IR radiation, the net change of dipole moment in molecule is
occurred and causes vibration of bonds in the molecule like Stretching and bending
vibration. The transmitted light is detected by the detector and IR spectrum
interpreted on the computer screen by analysis of the transmitted light.
 The IR spectroscopy theory utilizes the concept that molecules tend to absorb
specific frequencies of light that are characteristic of the corresponding structure of
the molecules. The energies are reliant on the shape of the molecular surfaces, the
associated vibronic coupling, and the mass corresponding to the atoms.
 For instance, the molecule can absorb the energy contained in the incident light and
the result is a faster rotation or a more pronounced vibration.
 INSTRUMENTATION
Fourier Transform Infra-Red Spectrophotometer
 FTIR stands for Fourier Transform Infra-Red Spectrophotometer — the preferred
method of infrared spectroscopy.
 A method for measuring all of the infrared frequencies simultaneously.
 FTIR testing identifies chemical compounds in consumer products, paints,
polymers, coatings, pharmaceuticals, foods and other products.
FTIR spectroscopy is preferred
 It is a non-destructive technique.
 It provides a precise measurement method which requires no external calibration.
 It can increase scan speed, collecting a scan every second.
Spectrometer components
Four basic components of FTIR system are
1. Radiation source
2. Sampling Techniques
3. Interferometer
4. Detector
[Link] sources:
 The most frequently used light source is the Globar, which is silicon carbide in form
of rods or helixes. A globar can be directly ignited, and has a burning temperature
of 1500 K, which needs to be water-cooled. The globar has the advantage of a
relatively high emissivity to about 100 cm-1, so it can be used into the far IR region.
Another source is the Nernst rod, which works at a temperature of 1900 K and is
used for the MIR region. It consists of zirconium oxide rods with additives of yttrium
oxides or oxides of other REE.
 The Nernst rod is a non-conductor at room temperature, initial heating is required
for ignition. It has following disadvantages: sensitive, deforms easily, oxide mix has
negative temperature coefficient of the electrical resistance, i.e. electrical
conductivity increases with temperature.
 Metallic helices, i.e., chromium nickel alloys or tungsten with operating
temperatures of about 1300K, are usually air-cooled and used in NIR. For NIR you
can also use tungsten-halogen lamps. For FIR, below 100 cm-1, you can
use mercury high-pressure lamps. It's plasma emission surpasses the spectral
radiation power of a black radiator of the same temperature.
 They must be started by a high-voltage pulse. Lasers can be used as radiation
sources, too. Gas lasers scan rather narrow spectral regions (CO 2 laser 1100 - 900
cm-1, CO laser 2000-1800 cm-1, NO2 laser 900-910 cm-1, He-Ne laser (632, 1.152
and 3.391 µm)). Semi-conductor diode lasers are larger tunable, and can cover
almost the whole MIR region, e.g., PbSnSe-type lasers. A disadvantage is their low
operation temperature of 15 - 90 K. Other examples, such as the gallium arsenide -
, or gallium aluminium arsenide lasers work in the IR region, and can be operated
at room temperature.
 For IR these are broad brand/continuous sources.
 Mostly they are just heat up metal or ceramics that emit IR light.
 Infrared sources consist of an inert solid that is electrically heated to a temperature
between 1,500 and 2,200K.
There are 4 types of radiation sources
1. Incandescent lamp
2. Nernst glower
3. Globar source
4. High pressure mercury arc
Incandescent lamp:
 In the near IR instruments an ordinary incandescent lamp is generally used.
 This fails in the far IR, because it is glass enclosed and has low spectral formation.
Nernst Glower:
 The Nernst glower is constructed of rare earth oxides in the form of a hollow
cylinder (zirconium, thorium).
 It is non conducting at room temperature and must be heated by external charge
to bring it to a conducting state.
 Glower is generally heated to a temperature between 1000 to 1800 Oc.
 Maximum radiation is about 7100cm-1
 Disadvantage of Nernst glower is frequent mechanical failure.
Globar Source:
 It is a rod of silicon carbide (5mm diameter, 50mm long) which is electrically
heated to about 1500K.
 It is heated to a temperature between 1300 and 1700 Oc.
 It emits maximum radiation at 5200cm -1.
 The main disadvantage is, it is a less intense source than the Nernst glower.
High pressure mercury arc:
 Used for Far IR region.
 It is a high pressure mercury arc which consist of a quartz – jacketed tube
containing Hg vapor.
 When current passes through the lamp, mercury is vaporized, excited, and
ionized, forming a IR radiation discharge at high pressure.
2. Sampling techniques: Sample cell & Sampling of substance:
Samples: liquids, gases, solids
 KBr pellets can can be used as sample holders or to dilute sample material. They
can be purchased (e.g., most common 13 mm in diameter) or produced with a
pressing tool. KBr is permeable to 400 cm-1. However, KBR is hygroscopic, so it
should be dried at 150 ºC and afterwards stored in an exsicator.
 Other usable materials are for example NaCl (to 650 cm-1), KI and CsI (to 200 cm-1).
 Pellet production: Alkali halogenides are able to cold-flow at pressures of ~ 0.7 " 1
GPa, the material singers and transparent tablets can be formed. To produce a tablet
in general 0.5 to 1.5 mg sample material is finely ground in an agate mortar; about
300 mg KBr powder are added and carefully mixed. The mixture is placed into the
press mould, the upper stamp precompresses the powder, which becomes equally
distributed. The pressing tool is inserted into a hydraulic press (with vacuum
pump). The assembly is exposed to 0.75 GPa (105 N) and the end pressure is held
for 2-10 minutes.
 Alternatively, one can prepare solutions, or oil suspensions, apply a layer or film on
a sample holder, or use the ATR method for insoluble materials.
Single-crystals, which are measured under the microscope, should have a double-
sided polish. The quality of the single-crystal preparation is especially important for
quantitative studies, in which the sample thickness is a parameter. Depending on
the measured species concentration you might have to thin the sample down to
avoid saturation of peaks.
 Additionally, samples can be placed into diamond-anvil cells or heating-cooling
stages, to obtain pressure- or temperature-dependent spectra, which might deliver
 useful additional information. Phase changes can occur, fine structure can become
observable, bands can split or shift, or in general a better spectral resolution might
be obtained.
Samples in Infrared Spectroscopy
The samples used in IR spectroscopy can be either in the solid, liquid, or gaseous state.
 Solid samples can be prepared by crushing the sample with a mulling agent which
has an oily texture. A thin layer of this mull can now be applied on a salt plate to
be measured.
 Liquid samples are generally kept between two salt plates and measured since the
plates are transparent to IR light. Salt plates can be made up of sodium chloride,
calcium fluoride, or even potassium bromide.
 Since the concentration of gaseous samples can be in parts per million, the sample
cell must have a relatively long pathlength, i.e. light must travel for a relatively long
distance in the sample cell.
Thus, samples of multiple physical states can be used in Infrared Spectroscopy.
 Infrared spectra may be obtained for Gases, Liquids, and Solids.
 Material containing sample must be transparent to the IR radiation. So the salts
like NaCl, KBr are only used.
Sample Cells:
 Attenuated Total Reflection (Atr)
 Transmission
Attenuated Total Reflection (ATR)
Use single bounce for pure samples, or high concentrations samples
 Use multi bounce for low concentration samples
 Multibounce ATRs give greater absorbance that single bounce ATRs.
Why does multibounce result in more absorbance than single bounce?
 Beer's law: A = ε b C
 b is the pathlength of the light passing through the sample
 more bounces means the light is interacting with sample more: effectively
increasing the pathlength

Solids:
 Typically you embed your powder (solid) into a matrix of KBr (potassium bromide).
 KBr pellets, discs – 13mm diameter, 0.3 mm thickness, hydraulic pressure 10 tons
load
Liquids:
 Thin film squeezed between 2 IR transparent windows and the thickness is 0.1 – 0.3
mm.
 Must be IR transparent
 Not soluble by the solvent
Gas Sample holder:
 Gas cells are usually 10 cm long for single pass transmission.
3. Michelson Interferometer:
 The heart of the FTIR is a Michelson Interferometer.
 The monochromator is replaced by an interferometer, which divides radiant beam,
then recombines them in order to produce repetitive interference signals measured
as a function of optical path difference as the name implies the interferometer
produces interference signals, which contain IR spectral information generated after
passing through a sample.
Consists of three active components:
1. A moving mirror
2. A fixed mirror
3. A beam splitter
Working:
 Radiation from the broadband IR source is collimated and directed into the
interferometer, and impinges on the beam- splitter.
 At the beam-splitter half of the IR beam is transmitted to the fixed mirror and the
remaining half is reflected to the moving mirror.
 After the divided beams are reflected from the two mirrors, they are recombined at
the beam-splitter.
 Due to changes in the relative position of the moving mirror, an interference pattern
is generated. The resulting beam then passes through the sample and is eventually
focused on the detector.

[Link] in IR: Detectors convert the optical signal into electrical signals. There's a
variety of different types, such as thermal, pneumatic, pyroelectric or photoelectric
detectors. Nowadays most detectors are photoelectric detectors because of their higher
sensitivity. Incident light alters the electrical conductivity in an irradiated semiconductor
material. The photosignal is measured as a change in voltage via the resistance or current.
Common types
InSb: photoconductive or photodiode, 10000 " 1500 cm-1, liquid-nitrogen- cooled
MCT: photoconductive, spectral range 12500 " 400 cm -1, liquid-nitrogen- cooled
DTGS: pyroelectric, MIR region, works at room temperature
Two most popular detectors for FTIR spectrometer are
1. Pyroelectric Detector: Deuterated triglycine sulphate (DTGS).
2. Photon-sensitive semiconducting Detectors: Mercury cadmium telluride (MCT)
DTGS: Room temperature detector, Less Expensive, Slow scanning.
MCT:
 It is a Cryogenic detector, More Expensive, fast scanning, interaction between
incident photon and semiconductor depends on quantum nature of radiation and
also exhibits very fast responses,
 It must be maintained at liquid nitrogen temperature to be effective.
 In general the MCT detector is faster and more sensitive than DTGS detector.

Application of IR Spectroscopy
 IR spectroscopy has been extensively used for qualitative as well as quantitative
analysis in academic labs as well as in industry. The extent of application of IR
spectroscopy for achieving a deeper insight of structural analysis and interactions
at molecular level has been intensive with the instrumental advancements.
 Concisely, IR spectroscopy is a promising tool in
 (1) establishing the chemical structure of small molecules, natural products, and
other biomolecules;
(2) identification of functional groups in sample; and
(3) identification and characterization of supramolecular interactions and chemical
bonding in supramolecular chemistry.
Forensic Applications:
i. Identification of an organic compound
ii. Structure determinations
iii. Identification of functional group
iv. Studying the progress of reaction
v. Determination of impurities in compound
1. Paints
2. Polymers
3. Coatings
4. Contaminants/Adulteration
5. Explosive residues
6. Narcotic Drugs
Limitations of IR Spectroscopy
a. Can’t determine the molecular weight of the compound.
b. Doesn’t give information about the relative position of different functional groups in
a molecule.
c. From the single IR spectra of an unknown substance, it is not possible to know
whether it is pure compound or a mixture of compound.
d. Sample cells are made of halogen salts which are susceptible to moisture
RAMAN SPECTROSCOPY
 Raman spectroscopy studies the inelastic scattering of light. The Raman effect was
predicted as early as 1923 by Adolf Smekal. In practice it was observed in 1928 by
the Indian scientist Sir Chandrasekhara Venkata Raman (physics Nobel price in
1930) in liquids and independently by Grigory Landsberg and Leonid Mandelstam
in crystals. In contrast to IR spectroscopy a change of the polarization potential, i.e.,
deformation of the electron cloud, is necessary for a molecule to exhibit a Raman
effect.
 The intensity of the scattered light is dependent on the amount of the polarization
potential change. As opposed to Brillouin spectroscopy, which studies elastic
properties of materials, photons are scattered by the interaction with vibrational
and rotational transitions in molecules. Raman spectroscopy is used to study a
material's chemical composition. Further applications are:
 Mineral Identification and Structural Characterization
 Analyses Of Gemstones and Archaeometry Objects
 Mineral Inclusions
 Speciation & Concentration
 Characterization Of E.G., Thermal Maturity, Metamictization, Oh Content,
Impurities
 Advantages of Raman spectroscopy are its non-destructive nature, small sample
amounts can be studied and no sample preparation is necessary.
PRINCIPLE / THEORY:
 When monochromatic radiation is incident upon a sample then this light will
interact with the sample in some fashion. It may be reflected, absorbed or scattered
in some manner. It is the scattering of the radiation that occurs which gives
information about molecular structure.
 Raman is based on scattering. The sample is irradiated with a coherent source,
typically a laser. Most of the radiation is elastically scattered (called the Rayleigh
scatter).A small portion is inelastically scattered (Raman scatter, composed of
Stokes and anti-Stokes portions).
 At temperatures higher than absolute zero all matter vibrates. A molecule can be
transferred to an excited state (vibration) through the absorption of a photon. The
energy has to be equal to the energy difference between the two vibrational states
(see IR spectroscopy - frequency principle). However, in Raman spectroscopy UV,
VIS or NIR light is used as radiation source, which has a much higher energy than
those energy differences and absorption of photons is impossible. Instead the
incident light will excite the system to a high-energy state.
 When it recovers from this state (immediately), scattering reactions occur. The
elastically scattered light has the same energy as the incident light - Rayleigh
scattering. If the system gains energy during this process, the scattered light loses
this amount of energy and the system reaches a higher energy state (higher energy
level) than it had before - Stokes scattering. Of an already vibrating system is
excited, it loses energy during this process and the system reaches a lower energy
level than it had before - Anti-Stokes scattering. The latter process is much more
rare, since at room temperature most molecules are in the ground state as opposed
to a higher energy level.
 The polarizability is a measure for the electron cloud's ability to deform in contrast
to the atomic nuclei. When placed into an electric (or oscillating) field the electrons
are pulled towards the positive charge and the atomic nuclei towards the negative
charge, which induces a dipole moment and results in scattering reactions. The
change of the polarizablity depends on the molecule geometry.
 For example, during the symmetric stretching vibration of the linear CO2 molecule
the polarizablitiy gets smaller during the stretching as opposed to the compression.
It changes, and the vibration is Raman-active (but IR-inactive). During the
asymmetric stretching vibration on the other hand the polarizability does not
change, and the vibration is Raman-inactive (but IR-active).
RAMAN SPECTRUM
 A Raman spectrum plots light intensity (unit, e.g., counts, counts per second or
arbitrary units) versus light frequency (relative wavenumbers). A Raman spectrum
consists of three parts, the intense Rayleigh line and less intense Raman bands in
the Stokes (red shifted, low-energy) and Anti-Stokes (blue shifted, high-energy) parts
of the spectrum, whereby the latter two parts are equal in energy.
 Generally, only the Stokes bands are recorded, because they are more easily
detectable due to their higher intensity. The Rayleigh line equals 0 Raman shift, so
that Anti-Stokes lines have negative wavenumbers and Stokes lines have positive
wavenumbers. The wavenumber shift is characteristic for a material.

INSTRUMENTATION
 The main components of a Raman system are a
1) Light source,
2) Optical components, such as lenses and mirrors, to focus the light onto a
sample and collect the scattered light,
3) A spectrometer,
4) A detector.
 The light source is typically a UV, VIS, or NIR laser emitting monochromatic light.
Types of lasers are gas lasers (e.g., Ar+, diode-pumped solid state lasers, or tunable
lasers. Notch filters are used to filter the Rayleigh line intensity before the scattered
light is entering the spectrometer and the detector (CCD camera).
 In the majority of experiments dispersive spectrometers are used (see IR section for
details) in combination with back-scatter, forward scatter or 90º geometries.
Depending on the instrumental setup, spectral resolutions of ~ 0.05 cm -1, depth
resolutions of about 2 µm and effective lateral resolutions of 1 " 1.5 µm can be
reached.
 Raman micro spectroscopy often takes advantage of a confocal setup to increase the
spectral resolution, where two apertures (behind the light source and before the
spectrometer) reduce stray light and eliminate out-of focus information and only
information from the focal plane reaches the detector.
Light Source:
 The sources used in modern Raman spectrometry are nearly always lasers because
their high intensity is necessary to produce Raman scattering of sufficient intensity
to be measured with a reasonable signal-to-noise ratio.
  Because the intensity of Raman scattering varies as the fourth power of the
frequency, argon and krypton ion sources that emit in the blue and green region of
the spectrum have and advantage over the other sources.

Sample Illumination System

 Liquid Samples: A major advantage of sample handling in Raman spectroscopy
compared with infrared arises because water is a weak Raman scattered but a
strong absorber of infrared radiation. Thus, aqueous solutions can be studied by
Raman spectroscopy but not by infrared.
 This advantage is particularly important for biological and inorganic systems and in
studies dealing with water pollution problems.
 Solid Samples: Raman spectra of solid samples are often acquired by filling a small
cavity with the sample after it has been ground to a fine powder. Polymers can
usually be examined directly with no sample pretreatment.
 Gas samples: Gas are normally contain in glass tubes, 1-2 cm in diameter and
about 1mm thick. Gases can also be sealed in small capillary tubes

FORENSIC APPLICATIONS:
 In forensic science, evidence such as biological fluids, drugs, and explosives is often
encountered and is of immense significance.
 Also, evidence such as paints, pigments, dyes, gunshot residue, and even fibers and
soil are commonly encountered.
 The use of RS in such cases allows the facile, rapid, and accurate determination of
the composition of the evidence.
 Non-destructive Narcotic Drug Identification
 Explosives Identification: Exact Chemical Compositions of Material (i.e. PETN, RDX)
Binding Agents Within Explosive
  Materials Identification and Analysis of Toxic Solvents and Bio-warfare Agents,
Trace Forensic Evidence Analysis: Including Fibers, Fabrics, Pigments, Inks,etc
Raman Spectra of Inorganic Species.
 The Raman technique is often superior to infrared for spectroscopy investigating
inorganic systems because aqueous solutions can be employed.
 In addition, the vibrational energies of metal-ligand bonds are generally in the range
of 100 to 700 cm-1, a region of the infrared that is experimentally difficult to study.
 These vibrations are frequently Raman active, however, and peaks with values in
this range are readily observed.
 Raman studies are potentially useful sources of information concerning the
composition, structure, and stability of coordination compounds.
Raman Spectra of Organic Species.
 Raman spectra are similar to infrared spectra in that they have regions that are
useful for functional group detection and fingerprint regions that permit the
identification of specific compounds.
 Raman spectra yield more information about certain types of organic compounds
than do their infrared counterparts.
 In addition, Raman sampling devices are not subject to attack by moisture, and
small amounts of water in a sample do not interfere.
 Biological Applications of Raman Spectroscopy Raman spectroscopy has been
applied widely for the study of biological systems.
 The advantages of his technique include the small sample requirement, the minimal
sensitivity toward interference by water, the spectral detail, and the conformational
and environmental sensitivity.

IR VS RAMAN SPECTRA
 The key difference between IR and Raman spectra is that IR spectra can be obtained
from light absorption, whereas Raman spectra can be obtained from light scattering.
 IR and Raman spectra are important in analytical chemistry for the determination
of light-absorbing and light scattering properties of different molecules.
 IR and Raman spectra are important in analytical chemistry for the determination
of light absorbing and light scattering properties of different molecules. The key
difference between IR and Raman spectra is that we can obtain the IR spectra from
light absorption and Raman spectra from light scattering.
 IR and Raman spectra are important in analytical chemistry for the determination
of light-absorbing and light scattering properties of different molecules. The key
difference between IR and Raman spectra is that IR spectra can be obtained from
light absorption whereas Raman spectra can be obtained from light scattering.
Besides, Raman spectra is a highly expensive method compared to IR.
IR Spectra:
 IR spectra or IR spectrum is the result of IR spectroscopy, where IR radiation is used
to analyze a sample. Here, we can observe the interaction between matter and IR
radiation. We can get IR spectra from absorption spectroscopy. IR spectroscopy is
used for the identification and analysis of chemical substances in a given sample.
Here, the sample can be solid, liquid or a gas. The instrument we can use to get an
IR spectrum is the infrared spectrophotometer.
 The IR spectrum is a graph. It has an absorbance of light by the sample in the y-
axis and wavelength or the frequency of IR light in the x-axis. The units of frequency
that we are using here is reciprocal centimetres (per centimetre or cm -1). If we are
using the wavelength instead of frequency, then the unit of measurement is
micrometres.
 An IR spectrum exploits the absorbance of different frequencies in IR radiation by
the molecules in a sample and the characteristics features of the chemical
structures. This is because the absorbed frequency of IR radiation is usually similar
to the vibrational frequency of the analyte molecule. We can get the IR spectra for
different molecules by passing a beam of IR radiation through the sample and
detecting the transmitted light through the sample. It gives us details about the
absorbed frequencies. Therefore, a typical IR spectrum is an absorption spectrum.

Raman Spectra:
 Raman spectra or Raman spectrum is an analytical technique that lies on the
inelastic scattering of photons in the sample. The inelastic scattering is called
Raman scattering. This technique is very useful in determining the vibrational
modes of molecules. Therefore, the Raman scattering effect is helpful in analytical
chemistry for providing a structural fingerprint by which we can identify different
molecules.
 The radiation we can use in the detection of a Raman spectra include visible, near
IR, or near UV range laser beams. However, near X-ray light beams can also be used
here. In this process, the laser beam reacts with the molecular vibrations
or phonons, resulting in the energy of the laser photons being shifted up or down.
 ATOMIC ABSORPTION SPECTROMETRY
 Atomic absorption spectroscopy, often abbreviated AAS, is the process which tests
the concentration of gas-phase atoms within a given sample. The concentration of
these atoms is determined by testing the amount of light absorbed by the free ions
within the sample. By exposing a sample to light at a specific wavelength and
tracking how much of that light is absorbed by the sample, scientists are not only
able to determine an element’s presence within a sample, but also that element’s
concentration.
 Atomic absorption spectroscopy can detect roughly 70 different elements and can
be utilized in both solid and liquid samples; though, the experimentation of solid
samples does require additional processes. Atomic absorption spectroscopy is
utilized across many industries and is instrumental in the detection of metals within
a sample. As such, this process is commonly utilized in pharmacology, archaeology,
manufacturing, mining, and forensics.
 Introduced in 1954 by Alan Walsh in Australia, Used for mining, medical treatment
& agriculture. To measure concentration by detecting absorption of electromagnetic
radiation by atoms. It is a very common technique for detecting metals in sample.
 Widely used in clinical laboratories to measure elements, Such as aluminum,
calcium, copper, lead, lithium, magnesium, zinc, and other metals
PRINCIPLE OF ATOMIC ABSORPTION SPECTROMETRY
 The basic principles of AAS can be expressed as follows. Firstly, all atoms or ions
can absorb light at specific, unique wavelengths. When a sample containing copper
(Cu) and nickel (Ni), for example, is exposed to light at the characteristic wavelength
of Cu, then only the Cu atoms or ions will absorb this light. The amount of light
absorbed at this wavelength is directly proportional to the concentration of the
absorbing ions or atoms.
 The electrons within an atom exist at various energy levels. When the atom is
exposed to its own unique wavelength, it can absorb the energy (photons) and
electrons move from a ground state to excited states. The radiant energy absorbed
by the electrons is directly related to the transition that occurs during this process.
Furthermore, since the electronic structure of every element is unique, the radiation
absorbed represents a unique property of each individual element and it can be
measured.
 Atomic absorption is an absorption spectrophotometric technique in which a
metallic atom in the sample absorbs light of a specific wavelength. The atoms absorb
ultraviolet or visible light and make transitions to higher electronic energy levels.
The analyte concentration is determined from the amount of absorption.
 Atomic absorption is a very common technique for detecting metals and metalloids
in environmental samples.
 INSTRUMENTATION
 Atomic Absorption Spectroscopy uses the absorption of light to measure the
concentration of gaseous atoms. The vaporization of the analyte ions or atoms is
carried out in flame or graphite furnace as the samples are generally solids or
liquids. Transitions to higher electronic energy level are made by the atoms as they
absorb visible or ultra-violet light.
 Determination of the analyte concentration is carried out by studying the amount
of absorption. In this process, a blank solution is sprayed into the flame and the
meter is adjusted for zero absorbance or 100% transmittance. Following this, the
solution under investigation is sprayed, the atoms in ground state absorb certain
part of light resulting decrease in transmitted light or increase in absorbed light
falling on photomultiplier tube. The readings can be tabulated and with the help of
standard graph, the concentration of a particular element in the sample can be
found out.
1. Light Source
2. Nebulizer
3. Atomizer
4. Monochromator
5. Detector
6. Amplifier
7. Readout device
1. RADIATION/LIGHT SOURCE
 The common source of light is a Hollow Cathode Lamp (HCL). This contains a
tungsten anode and a cylindrical hollow cathode made of the element to be
determined. These are sealed in a glass tube filled with an inert gas e.g., neon or
argon at a pressure of between 1 N/m2 and 5 N/m2. The ionisation of some gas
atoms occurs by applying a potential difference of about 300 – 400 V between the
anode and the cathode.
 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 characteristic of the metal as they fall back to the ground state. These
emitted radiations form incident radiations for the element under analysis.
 Hollow Cathode Lamp are the most common radiation source in AAS. 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.
2. NEBULIZER IN ATOMIC ABSORPTION SPECTROMETRY
 Suck up liquid samples at controlled rate.
 Create a fine aerosol spray for introduction into flame.
 Mix the aerosol and fuel and oxidant thoroughly for introduction into flame.
ATOMIZER IN ATOMIC ABSORPTION SPECTROMETRY
Atomizer: Nebulisation is the mechanism by which the sample solution is introduced as
fine spray into the flame. Nebulisation refers to the dispersion of a liquid into particles by
a rapidly moving gas, liquid stream or by mechanical means. This process is immediately
followed by atomization, wherein high energy like that of a flame converts molecules into
atoms.
 Elements to be analyzed needs to be in atomic sate.
 Atomization is 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.
 Atomizer converts the liquid into small droplets which are easily vaporized.

FLAME ATOMIZER:
 The burner and a nebulizer help in the atomization of the element. Atomization
occurs in the flame and an atomic vapour of the element to be analysed is produced.
The selection of flame temperature is important for atomization. When it is low,
atomization will be partial and when it is high, the atoms may get ionized.
 Flame atomizers, frequently abbreviated FAAS, are the oldest and most commonly
used atomizers in atomic absorption spectroscopy. FAAS is most commonly used to
test liquid samples or solid samples which have been dissolved within a liquid. Using
this atomization process, a sample is first evaporated, leaving behind only the
sample’s dry nano-particles.
 These solid particles are then vaporized and converted into gaseous molecules,
which can then be dissociated into free atoms. Finally, the atoms are converted into
gaseous ions and can be exposed to a small flame, which can reach extremely high
 temperatures. It is during this step in the process that the sample is finally exposed
to a radiation beam and the signal of absorbed electromagnetic radiation is
measured.
 To create flame, we need to mix an oxidant gas and a fuel gas. In most of the cases
air-acetylene flame or nitrous oxide- acetylene flame is used. Liquid or dissolved
samples are typically used with flame atomizer.
GRAPHITE TUBE ATOMIZER:
 Electrothermal atomizers are sometimes also referred to as graphite furnace
atomizers, as they utilize a graphite tube to heat samples, rather than a flame.
Unlike flame atomization, which transforms the sample solution into an aerosol and
mixes it with flame gases, this technique allows liquid, solid, and gaseous samples
to be analyzed directly.
 Electrothermal/graphite furnace atomizers, sometimes abbreviated ETAAS or
GFAAS, deliver signals in a discontinuous mode. By contrast, flame atomizers
deliver signals in a continuous fashion. ETAAS/GFAAS also minimize interference
problems and can determine a wide variety of elements for most matrices.
 Uses a graphite coated furnace to vaporize the sample. ln GFAAS sample, samples
are deposited in a small graphite coated tube which can then be heated to vaporize
and atomize the analyte. The graphite tubes are heated using a high current power
supply.
 A graphite furnace can be employed instead of flame for atomization. The atomizer
may be elongated along its axis to increase the distance between the optical path
and the sample deposition point which increases the analytical sensitivity.

MONOCHROMATORS AND FILTERS USED IN AAS:

Monochromator: It is important that the instrument be capable of providing a narrow
band width to separate the line chosen for determination from other undesirable lines.
Usually used devices are gratings or prisms.
 In simple flame photometers, the monochromators are the prism.
 QUARTZ is the material most commonly used for making prisms because quartz is
transparent over entire region.
 FILTERS: The filter is made up of such material which is transparent over a
narrow spectral range.
 When a filter is kept between the flame detector, the radiation of the desired
wavelength from the flame will be entering the detector and be measured.
 The remaining undesired wavelength will be absorbed by the filter and not
measured.
 In flame photometry, the wavelength as well as intensity of radiation emitted by
the element has to be monitored. Hence a filter or monochromator is used.
DETECTORS:
Photomultiplier tube:
 Photo multiplier tube Photo multiplier tube is generally used in spectroscopy. It
consists of an evacuated tube containing one photocathode and 9 to 16 electrodes
known as dynodes.
 Photocathode is negatively charged and then a photon hits the photocathode.
Emission of electrodes takes place due to photovoltaic effect. When incident
radiation falls on the metal surface of cathode and get emitted which are attracted
towards the first dynode.
 These with are attracted by the second dynode and with are emitted by second
dynode. Hence, the process is repeated at all the dynodes. At the end of dynode
chain and anode is present which acts as collector of electrons.
 The current flowing from anode is directly proportional to photo electron flux
generated by photo cathode. Ejected photoelectron strikes dynode, secondary
electrons released, voltage accelerates secondary electrons to next dynode and so
on, then big voltage divider. Result is large charge packet hitting anode.
 3. Photovoltaic cell:
 It consists of semi-conductor like selenium which is deposited on a strong metal
base such as iron. A thin layer of gold or silver is sprayed over the semiconductor
which then acts as second collective electrodes. When radiations fall on the
surface, it produces electrons at the selenium silver interface. Since, barriers
exist between selenium and iron. The flow of electron is prevented which results
in accumulation of electron on the silver surface and produces electrical voltage.
 The Photovoltaic cell is the semiconductor device that converts the light into
electrical energy. The voltage induces by the PV cell depends on the intensity of
light incident on it. The name Photovoltaic is because of their voltage producing
capability.
 The electrons of the semiconductor material are joined together by the covalent
bond. The electromagnetic radiations are made of small energy particles called
photons. When the photons are incident on the semiconductor material, then the
electrons become energised and starts emitting.
 The energises electron is known as the Photoelectrons. And the phenomenon of
emission of electrons is known as the photoelectric effect. The working of the
Photovoltaic cell depends on the photoelectric effect.

Amplifier:
It amplifies the signal from the detector which is received by the read-out.
Readout:
It reads the data and gives the information regarding the metals, qualitatively &
quantitatively.
FORENSIC APPLICATIONS:
 Atomic absorption spectroscopy has been utilized in the study of forensic sciences
for many years. Using this technology, forensic scientists can perform in-depth
analysis of blood samples, brain and muscle tissue, and gunshot powder residue.
 This technology has vastly improved the accuracy of toxicology reports in cases of
metal poisoning. Common causes of metal poisoning, such as mercury and lead,
are easily detectable using this technology and can be identified even in trace
amounts.
 Atomic absorption spectroscopy (AAS) has found wide application as a bulk analysis
technique for GSR. This method of analysis is capable of sequentially determining
lead, antimony, and barium; however, in order to detect the low concentration of
these species that may be present in GSR, the more sensitive flameless AAS
techniques such as electrothermal atomization are required. More
recently, inductively coupled plasma atomic emission spectroscopy has also been
applied to the analysis of GSR; however, it has not found widespread use,
presumably due to the high cost of the instrumentation.
  Determination of even small amounts of metals (Lead, Mercury, Calcium,
Magnesium etc.)
 Environmental studies: Drinking water, Ocean Water, Soil Analysis, petrol etc.
 Analysing metals in biological fluids such as blood urine, viscera samples etc.
 Trace elemental analysis in cosmetics, hair, nails etc.
 Atomic absorption spectroscopy is frequently utilized in agriculture and the study
of environmental sciences. Atomic absorption spectroscopy, as well as atomic
fluorescence spectroscopy—which analyzes the light emitted from a sample rather
than the light absorbed—are frequently used in various fields of agricultural study.
Typically, they’re used to identify and analyze the presence of potentially harmful
elements in the environment.
 One common use for these methods is the analysis of soil samples and the effect
the quality of the soil will have on the overall rate of food production in a certain
area. Soil samples that contain high levels of phosphorous and nitrogen often yield
higher production volume and produce healthier crops. Atomic absorption and
atomic fluorescence spectroscopy can be used to determine the presence of these
elements and the quantities in which they appear. These methods can also be used
to detect trace amounts of harmful chemicals, such as rhodium, in water samples.
 Atomic absorption spectrometry has many uses in different areas of chemistry. The
different methods of Atomic absorption spectrometry are very powerful for analysis
elements in a solution.
 The instruments are simple and easy to operate. They are useful when few elements
have to be determined in a large number of samples, as is the case in clinical or food
analysis.
 Atomic absorption spectroscopy methods are of great importance compared with
other methods of elemental analysis. In clinical analysis, it is used for analysing
metals in biological fluids such as blood and urine.
 In environmental analysis, it is utilized for monitoring the levels of various elements
in rivers, seawater, drinking water and air etc., Presence of contaminants can be
monitored in food stuff like fruit juices and wines.
 AAS can trace presence of pesticide residues in fruits and vegetables. In
pharmaceuticals industries, it is used for the assay of drugs. Purity of the sample
can be checked for minute quantities of a catalyst used in the manufacturing
process (usually a metal) sometimes present in the final product. Levels of the toxic
substances present in the products can be verified and reduced by this technique.
By using Atomic absorption spectrometry in mining industries, 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.
ADVANTAGES AND DISADVANTAGES OF ATOMIC ABSORPTION SPECTROMETRY
Advantages
1. High selectivity and sensitivity
2. Fast and simple working
3. Doesn’t need metals separation
Disadvantages
1. Analysis doesn’t simultaneous
2. Fragment have to form ready measure solution
3. Limit types of cathode lamp (expensive)
4. The efficiency of atomization is poor.
5. Poor sensitivity
 INTRODUCTION TO FLAME EMISSION SPECTROMETRY
 During 1980s Bowling Barnes, David Richardson, John Berry and Robert Hood
developed an instrument to measure the low concentrations of sodium and
potassium in a solution.
 They named this instrument as Flame photometer also known as Flame Emission
Spectroscopy
 To measure emission of characteristic radiation in flames by
individual elements and the correlation of the emission intensity with the
concentration of these elements.
PRINCIPLE OF FLAME EMISSION SPECTROMETRY
 The principle of flame photometer is based on the measurement of the emitted light
intensity when a metal is introduced into the flame.
 The wavelength of the colour gives information about the element and the colour of
the flame gives information about the amount of the element present in the sample.
 Some of these atoms further get excited to even higher levels. But these atoms are
not stable at higher levels.
 Hence, these atoms emit radiations when returning back to the ground state. These
radiations generally lie in the visible region of the spectrum. Each of the alkali and
alkaline earth metals has a specific wavelength.
 In flame emission spectroscopy, the combustion flame does not only free the atom
it also supplies the energy necessary to move the electrons of the free atoms from
ground state to excited state. The energy which is emitted by the excited atoms when
returning to the ground state provides the basis for analytical determination in FES.
 External source (like hallow cathode lamp) is used to excite the atoms from its
ground state. • The flame that contains the free atom becomes the sample cell. The
free atoms absorb radiation focused on the cell from external source. • Incident
radiation absorbed by the atoms moving from ground state to the excited state
provides analytical data.
 INSTRUMENTATION
The basic components for flame photometer are as follows:
1. Burner
2. Atomizer
3. Monochromators
4. Detectors
5. Amplifier
6. Readout device
[Link]:
The flame used in the flame photometer should possess following functions:
 The flame should have ability to evaporate the liquid droplets from the sample
solution in the formation of solid residue.
 The flame should decompose the compounds in the solid residue resulting in the
formation of atoms.
 The flame must have the capacity to excite the atoms formed and cause them to
emit radiant energy.

TYPES OF BURNERS:
1. Mecker Burner
2. Total Consumption Burner
3. Laminar Flow Burner
4. Lunder graph Burner
5. Shielded Burner
6. Nitrous Oxide – Acetylene Flame Burner
 This is a part that produces excited atoms. Here the sample solution is sprayed into
fuel and oxidant combination. A homogenous flame of stable intensity is produced.
Mecker Burner:
 This burner employed natural gas and oxygen.
 Produces relatively low temperature and low excitation energies.
 This are best used for alkali metals only.
 Nowadays it is not use.
Total Consumption Burner:
 In this burner the fuel and oxidant are hydrogen and oxygen gas respectively.
 In this the sample solution is aspirated through a capillary by the high pressure.
fuel and oxidant are burnt at the tip of the burner.
 The name “total consumption burner” is used because all the sample that enters
the capillary will enter the flame regardless of the droplet size.
Laminar Flow Burner:
 In this type of the burner, aspirated sample, fuel and oxidant are thoroughly mixed
before reaching the burner opening and then entering the flame.
 Important feature of this is that only a small portion (about 5%) of the sample
reaches the flame in the form of small droplets and is easily decompose.
Lundergraph Burner:
 In this, sample and air is mixed in a chamber, this mixed composition is send to
fuel nozzle where it is atomized.
 Here the sample reaches the flame is only about 5%.
Shielded Burner:
 In this flame was shielded from the ambient atmosphere by a stream of inert gas.
 Shielding is done to get better analytical sensitivity and quieter flame.
Nitrous oxide – Acetylene flame Burner:
 These flames were superior to other flames for effectively producing free atoms.
 The drawback of it is the high temperature reduces its usefulness for the
determination of alkali metals as they are easily ionized and Intense background
emission, which makes the measurement of metal emission very difficult NITROUS
OXIDE ACETYLENE FLAME.
ATOMIZER IN FLAME EMISSION SPECTROMETRY
 Process of conversion of sample to a fine mist of finely divided droplets using a jet
of compressed gas.
 It is conversion of molecules to their component atoms in gaseous state, and it is
carried out by introduction of the molecule’s solution in the flame in very fine
droplet.
Light Source: Hollow cathode lamps (HCL),
Nebulizer-burner system: The production of gaseous atoms is the primary objective of
the nebulizer-burner system. The flame used in the AFS must be able to evaporate the
liquid droplets and decompose the solid compounds leading to the formation of the
gaseous atom.
FORENSIC APPLICATIONS OF AFES:
 Clinical: Analysis of Pb, Hg, As, Sb, Bi, Ge, Se, in blood, urine, tissue, nail, hair; Cu,
Zn and Pb in blood serum and urine samples.
 Agricultural: Analysis of dairy, wine, feed, meat, cigarettes, and other products for
As, Hg, Pb, Sb, Se.
 Geological and Metallurgical: Analysis of ore, rock, mineral, metals for Ge, Hg, Se,
As in Sb, Se, Te in Cu.
 Pharmaceutical: Determination of Hg, Pb, As, Se in active ingredients and fillers.
 Petrochemical: Quantitative determination of Pb, Hg, Cd, As, Sn, Zn in fuels,
lubricant, crude oil.
ADVANTAGES OF FLAME FLAME EMISSION SPECTROMETRY
 The method of analysis is very simple, inexpensive, and more convenient.
 A simple method for both qualitative & quantitative analysis based on flame
analysis.
 It is quick, selective, and sensitive technique.
 Even very low concentrations (parts per million to parts per billion range) of metals
in the sample can be determined.
 This method compensates for any unexpected interfering material present in the
sample solution.
DIS ADVANTAGES OF FLAME FLAME EMISSION SPECTROMETRY
In spite of many advantages, this analysis technique has quite a few disadvantages:
 The accurate concentration of the metal ion in the solution cannot be measured.
 Though this technique measures the total metal content present in the sample, it
does not provide the information about the molecular structure of the metal present
in the sample.
 Only liquid samples may be used.
 Also sample preparation becomes lengthy in some cases.
 Flame photometry cannot be used for the direct determination of each and every
metal atom.
 A number of metal atoms cannot be analyzed by this method. The elements such as
carbon, hydrogen and halides cannot be detected due to their non-radiating nature.
MASS SPECTROSCOPY
 Mass spectrometry is an analytical tool useful for measuring the mass-to-charge
ratio (m/z) of one or more molecules present in a sample. These measurements can
often be used to calculate the exact molecular weight of the sample components as
well. Typically, mass spectrometers can be used to identify unknown compounds
via molecular weight determination, to quantify known compounds, and to
determine structure and chemical properties of molecules.
 An instrument that literally measures mass of molecules. Mass spectrometry (MS)
is an analytical chemistry technique that helps in identify the amount and type of
chemicals present in a sample by measuring the mass-to-charge ratio.
 It is a powerful technique for chemical analysis that is used to identify unknown
compounds, to quantify known compounds, and to elucidate molecular structure.
A mass spectrum is a plot of the ion signal as a function of the mass-to- charge
ratio.
How does a mass spectrometer perform such a feat? Every mass spectrometer consists of
at least these three components:
 Ionization Source
 Mass Analyzer
 Ion Detection System
 Mass spectrometry is an analytical method useful for calculating the mass-to-
charge ratio (m / z) of one or more molecules in the sample. Such measurements
may also often be used to determine the precise molecular weight of the sample
components. Mass spectrometry is an analytical method to find the molecular mass
of a compound and indirectly helped to prove the identity of isotopes.
PRINCIPLE/THEORY:
 A mass spectrometer generates multiple ions from the sample under
investigation, it then separates them according to their specific mass-to-
charge ratio (m/z), and then records the relative abundance of each ion
type.
 The first step in the mass spectrometric analysis of compounds is the
production of gas phase ions of the compound, basically by electron
ionization. This molecular ion undergoes fragmentation. Each primary
product ion derived from the molecular ion, in turn, undergoes
fragmentation, and so on.
 The ions are separated in the mass spectrometer according to their mass-
to-charge ratio, and are detected in proportion to their abundance. A mass
spectrum of the molecule is thus produced. It displays the result in the
form of a plot of ion abundance versus mass-to-charge ratio. Ions provide
information concerning the nature and the structure of their precursor
molecule. In the spectrum of a pure compound, the molecular ion, if
present, appears at the highest value of m/z (followed by ions containing
heavier isotopes) and gives the molecular mass of the compound.
 A mass spectrometer is a “Molecular Smasher”. In this technique, molecules are
bombarded with a beam of energetic electrons (70eV) in gaseous state under
pressure of Hg, using tungsten filament. Molecules are broken up into cations and
many other fragments.
 The molecules are ionized and broken up into many fragments, some of which are
positive ions. Each kind of ion has a particular ratio of mass to charge, i.e. m/e
ratio(value).
 The ions pass through magnetic and electric fields to reach detector where they are
detected and signals are recorded to give mass spectra. Based on Newton’s second
law of motion and momentum, a mass spectrometer uses this property of matter to
plot ions of varying masses on a mass spectrum. From the law, we infer how much
mass is relevant to the inertia and acceleration of a body. This principle is applied
to the aspect where ions with different mass to charge ratios are deflected by
different angles in an electric or magnetic field.
 The following describes the operation of a spectrometer mass analyzer, which is of
the sector type. (Other analyzer types are treated below.) Consider a sample
of sodium chloride (table salt). In the ion source, the sample is vaporized (turned
into gas) and ionized (transformed into electrically charged particles)
into sodium (Na+) and chloride (Cl−) ions.
 Sodium atoms and ions are monoisotopic, with a mass of about 23 u. Chloride
atoms and ions come in two stable isotopes with masses of approximately 35 u (at
a natural abundance of about 75 percent) and approximately 37 u (at a natural
abundance of about 25 percent). The analyzer part of the spectrometer
 contains electric and magnetic fields, which exert forces on ions traveling through
these fields.
 The speed of a charged particle may be increased or decreased while passing
through the electric field, and its direction may be altered by the magnetic field. The
magnitude of the deflection of the moving ion's trajectory depends on its mass-to-
charge ratio.
 Lighter ions get deflected by the magnetic force more than heavier ions (based
on Newton's second law of motion, F = ma). The streams of sorted ions pass from
the analyzer to the detector, which records the relative abundance of each ion type.
This information is used to determine the chemical element composition of the
original sample (i.e. that both sodium and chlorine are present in the sample) and
the isotopic composition of its constituents (the ratio of 35Cl to 37Cl).

INSTRUMENTATION
COMPONENTS OF A MASS SPECTROMETER
 SAMPLE INLET: To introduce very small amount of sample into the mass
spectrometer. Components are converted into gaseous ions.
 AN IONIZATION SOURCE: Convert the components of a sample into ions. Output
is a stream of positive or negative ions, Most of the positive ions formed will carry
charge of +1 that are then accelerated into mass analyser.
 A MASS ANALYZER: Ions are dispersed based on the mass-to-charge ratios of the
analyte ions.
 Detector/Data System: That converts the beam of ions into an electrical signal
that can then be processes, stored in the memory of a computer, and displayed or
recorded.
 1. Sample Inlet
2. Ionization source
3. Mass Analyser
4. Detector
5. Data System
IONIZATION SOURCE:
 Molecules are converted to gas-phase ions so that they can be moved about and
manipulated by external electric and magnetic fields. In our laboratory we use a
technique called nano electrospray ionization, which is somewhat similar to how
cars are industrially painted.
 This method allows for creating positively or negatively charged ions, depending on
the experimental requirements. Nano electrospray ionization can directly couple
the outlet of a small-scale chromatography column directly to the inlet of a mass
spectrometer. The flow from the column is passed through a needle that is 10-15
um at its tip.
 It converts components of a sample into ions by bombardment with electrons, ions,
molecules. An electron which sticks a molecule may impart enough energy to remove
another electron from that molecule. The charged molecule is known as molecular
ion. The molecular ion can cause that ion to break into smaller pieces.

ELECTRON IMPACT (EI)

 The most widely used and highly developed method. It is also known as electron
bombardment.
 Electrons are produced by tungsten filament
 These electrons accelerated towards the ion source chamber
 The electrons require an energy equal to the voltage between the filament & ion
source chamber
 70ev is commonly used
 A proportion of electron beam will strike the electron trap producing trap current.

CHEMICAL IONIZATION
 In chemical ionization the ionization of the analyte is achieved by interaction of its
molecules with ions of a reagent gas in the chamber or source.
 Chemical ionization is carried out in an instrument similar to electron impact ion
source with some modifications such as:
 Narrowing of exit slit to mass analyser to maintain reagent gas pressure of about 1
torr in the ionization chamber.
 Providing a gas inlet.
  CI is a two-part process.
STEP-1: Reagent gas is ionized by electron impact ionization in the source.
The primary ions of reagent gas react with additional gas to produce stabilized
reagent ions.
STEP-2: Reagent ions interact with sample molecules to form molecular ions.
 In this technique the sample is diluted with large excess of reagent gas. Gasses
commonly used as reagent are low molecular weight compounds such as methane,
tertiary isobutane, ammonia, nitrous oxide, oxygen and hydrogen etc.

ELECTRON SPRAY IONIZATION

 The method generates ions from solution of a sample by creating fine spray of
charged droplets.
 A solution of sample is pumped through a fine, charged stainless steel capillary
needle at rate of few microliters/minute.
 The needle is maintained at a high electrical field (several kilo volts) with respect to
cylindrical electrode.
 The liquid pushes itself out of the capillary as a mist or aerosol of fine charged
droplets.

MASS SELECTIVE ANALYZER

 Once ionized, the ions are sorted and separated according to mass-to-charge (m/z)
ratios. There are a number of mass analyzers currently available, each of which has
trade-offs relating to speed of operation, resolution of separation, and other
operational requirements. The specific types in use at the Broad Institute are
discussed in the next section. The mass analyzer often works in concert with the
ion detection system.
 Analyser is the section of instrument that separates ions of different m/z ratios.
They deflect ions down a curved tubes in a magnetic field based on their mass,
charge ratios.
  The magnetic field is scanned to measure different ions.
1. Quadra pole mass analyser
2. Ion trap mass analyser
3. Time of flight mass analyser

QUADRAPOLE MASS ANALYZER

 Also known as Hewlett-Packard” or Mass Selective Detector”. In quadrupole mass
analyser a set of four rods are arranged parallel to the direction. Only m/z is been
determined and stable oscillation takes place. Ions travels in quadrupole axis with
cork screw type of trajectory. It functions as a mass filter.
 Quadrupole mass analyzers use oscillating electrical fields to selectively stabilize or
destabilize the paths of ions passing through a radio
frequency (RF) quadrupole field created between four parallel rods. Only the ions in
a certain range of mass/charge ratio are passed through the system at any time,
but changes to the potentials on the rods allow a wide range of m/z values to be
swept rapidly, either continuously or in a succession of discrete hops.
 A quadrupole mass analyzer acts as a mass-selective filter and is closely related to
the quadrupole ion trap, particularly the linear quadrupole ion trap except that it is
designed to pass the untrapped ions rather than collect the trapped ones, and is for
that reason referred to as a transmission quadrupole. A magnetically enhanced
quadrupole mass analyzer includes the addition of a magnetic field, either applied
axially or transversely.
 This novel type of instrument leads to an additional performance enhancement in
terms of resolution and/or sensitivity depending upon the magnitude and
orientation of the applied magnetic field. A common variation of the transmission
quadrupole is the triple quadrupole mass spectrometer. The “triple quad” has three
consecutive quadrupole stages, the first acting as a mass filter to transmit a
particular incoming ion to the second quadrupole, a collision chamber, wherein that
ion can be broken into fragments.
 The third quadrupole also acts as a mass filter, to transmit a particular fragment
ion to the detector. If a quadrupole is made to rapidly and repetitively cycle through
a range of mass filter settings, full spectra can be reported. Likewise, a triple quad
can be made to perform various scan types characteristic of tandem mass
spectrometry.
ION TRAP MASS ANALYZER
 The ion trap mass analyser operates by similar principles where it consists of
circular ring electrode and two end caps that form a chamber.
 AC or DC power along Radio Frequency potential is applied between the cups and
the ring electrode.
 The quadrupole ion trap works on the same physical principles as the quadrupole
mass analyzer, but the ions are trapped and sequentially ejected. Ions are trapped
in a mainly quadrupole RF field, in a space defined by a ring electrode (usually
connected to the main RF potential) between two endcap electrodes (typically
connected to DC or auxiliary AC potentials). The sample is ionized either internally
(e.g. with an electron or laser beam), or externally, in which case the ions are often
introduced through an aperture in an endcap electrode.
 There are many mass/charge separation and isolation methods but the most
commonly used is the mass instability mode in which the RF potential is ramped so
that the orbit of ions with a mass a > b are stable while ions with mass b become
unstable and are ejected on the z-axis onto a detector. There are also non-
destructive analysis methods.
 Ions may also be ejected by the resonance excitation method, whereby a
supplemental oscillatory excitation voltage is applied to the endcap electrodes, and
the trapping voltage amplitude and/or excitation voltage frequency is varied to bring
ions into a resonance condition in order of their mass/charge ratio
TIME OF FLIGHT MASS ANALYZER
 The time-of-flight (TOF) analyser uses an electric field to accelerate the ions through
the same potential. Then it measures the time take to reach the detector. If the
particle all have the same charge, the kinetic energies will be identical and their
velocities will depend upon only on their masses. Lighter ions travel faster and strike
the detector first so that the m/z ratio of ions is detected.
 The time-of-flight (TOF) analyzer uses an electric field to accelerate the ions through
the same potential, and then measures the time they take to reach the detector. If
the particles all have the same charge, their kinetic energies will be identical, and
their velocities will depend only on their masses. Ions with a lower mass will reach
the detector first.
 However, in reality, even particles with the same m/z can arrive at different times
at the detector, because they have different initial velocities. The initial velocity is
often not dependent on the mass of the ion, and will turn into a difference in the
final velocity. Because of this, ions with the same m/z ratio will reach the detector
at a variety of times, which broadens the peaks shown on the count vs m/z plot,
but will generally not change the central location of the peaks, since the starting
velocity of ions is generally centered at zero. To fix this problem, time-lag
focusing/delayed extraction has been coupled with TOF-MS

DETECTORS
 The separated ions are then measured and sent to a data system where
the m/z ratios are stored together along with their relative abundance. A mass
spectrum is simply the m/z ratios of the ions present in a sample plotted against
their intensities. Each peak in a mass spectrum shows a component of
unique m/z in the sample, and heights of the peaks connote the relative abundance
of the various components in the sample.
 Once the ions are separated by the mass analyser, they reach the ion detector, which
generates a current signal from the incident ions.
 The most commonly used detectors in MS are as follows:
1. Faraday cup
2. Electron multiplier
FARADAY CUP DETECTOR
 A faraday cup involves an ion striking the dynode surface which causes secondary
electrons to be ejected.
 This temporary electron emission induces a positive charge on the detector and
therefore a current of electrons flowing toward the detector.
 It measures the ion current hitting a metal cup, and is sometimes used for high
current secondary ion signals.
  The resulting current can be measured and used to determine the number of ions
or electrons hitting the cup.

ELECTRON MULTIPLIER DETECTOR

 It is the most common means of detecting ions. It is made up of a series (12 to 24)
of aluminium oxide dynodes maintained at ever increasing potentials.
 Ions strikes the first dynode surface causing an emission of electrons. These
electrons are then attracted to the next dynode held at a higher potential and
therefore more secondary electrons are generated.
 Ultimately as numerous dynodes are involved a cascade of electrons is formed that
results in an overall current gain on the order of one million or higher.

APPLICATIONS
 Environmental monitoring and analysis (Soil, water, air pollutants, water quality
etc.
 Chemical and petrochemical industry – quality control.
 Applications in biotechnology: identify structures of biomolecules, such as
carbohydrates, nucleic acids.
 Forensic (Arson, Explosives, Drugs, Unknown compounds)
 Pesticide analysis, food safety and quality
 Clinical toxicology
 Forensic toxicology
 Food, beverages and perfume analysis
 Petrochemical and hydrocarbon analysis
 INDUCTIVELY COUPLED PLASMA-MASS SPECTROSCOPY
 ICP Mass Spectrophotometers (ICP-MS) was first introduced by R.S. Houk, A.L. Gray
et al. in 1980. An analytical technique to determine elements using mass
spectrometry from ions generated by an inductively coupled plasma. Employs
plasma as ionization source and mass spectrometer as analyser for detection of ions.
 The Inductively Coupled Plasma (ICP) is an ionization source that fully decomposes
a sample into its constituent elements and transforms those elements into ions. It
is typically composed of argon gas, and energy is "coupled" to it using an induction
coil to form the plasma.
 It can perform both qualitative and quantitative analysis. It is used to
detect metals and several non-metals in liquid samples at very low concentrations
(PPM). Compared to atomic absorption spectroscopy, ICP-MS has greater speed,
precision, and sensitivity.
 Inductively coupled plasma (ICP) sources are used primarily for cation analysis of a
wide array of sample types. In this source, a plasma that is electrically neutral
overall, but that has had a substantial fraction of its atoms ionized by high
temperature, is used to atomize introduced sample molecules and to further strip
the outer electrons from those atoms.
 The plasma is usually generated from argon gas, since the first ionization energy of
argon atoms is higher than the first of any other elements except He, F and Ne, but
lower than the second ionization energy of all except the most electropositive metals.
The heating is achieved by a radio-frequency current passed through a coil
surrounding the plasma.
PRINCIPLE:
 The first stage of the interface consists of two cone-shaped openings: a sampler cone
and a skimmer cone. The hot plasma from the ICP torch enters the first stage of the
interface through the sampler cone, which is a pin-hole with a diameter of
approximately 1 mm. Samples in solution form are drawn directly into the ICP torch
using a nebulizer. Solid samples are vaporized using a laser (a process called laser
ablation) and the vapor drawn directly into the ICP torch.
 A pump is used to drop the pressure in the first stage to approximately 1 torr. The
expansion of the plasma as it enters the first stage results in some cooling of the
plasma. The skimmer cone allows a small portion of the plasma in the first stage to
pass into the second stage, which is held at the mass spectrometer's operating
pressure of approximately 10–5 torr. A series of ion lenses are used to narrow the
conical dispersion of the plasma, to isolate positive ions from electrons, neutral
species, and photons—all of which will generate a signal if they reach the
transducer—and to focus the ion beam onto the quadrupole's entrance.
 Liquid droplets are formed on the tip of a needle, where they become nebulized
due to argon gas flowing through a second needle perpendicular to the sample
needle. Aerosol passes into spray chamber where large droplets are removed via a
drain. Only 2% of original mist enters the spray chamber.
 Inductively coupled plasma (ICP) sources radio frequency energy to an Ar
gas stream. The RF energy completely ionizes the argon gas to generate a high-
temperature plasma that can effectively ionize elements with very high ionization
potentials.
 Sample aerosol decomposed in plasma (6000-10000K) to form analyte atoms
which are simultaneously ionized. Ions are directed into a mass filtering device
known as the mass spectrometer.
FORMATION OF PLASMA:
 To generate plasma, first, argon gas is supplied to torch coil, and high frequency
electric current is applied to the work coil at the tip of the torch tube. Using the
electromagnetic field created in the torch tube by the high frequency current, argon
gas is ionized and plasma is generated. This plasma has high electron density and
temperature (10000K) and this energy is used in the excitation-emission of the
sample. Solution samples are introduced into the plasma in an atomized state
through the narrow tube in the center of the torch tube.
 The plasma used in an ICP-MS is made by partially ionizing argon gas (Ar → Ar+ +
e−). The energy required for this reaction is obtained by pulsing an alternating
electric current in load coil that surrounds the plasma torch with a flow of argon
gas.
 After the sample is injected, the plasma's extreme temperature causes the sample
to separate into individual atoms (atomization). Next, the plasma ionizes these
atoms (M → M+ + e−) so that they can be detected by the mass spectrometer.
 An inductively coupled plasma (ICP) for spectrometry is sustained in a torch that
consists of three concentric tubes, usually made of quartz. The two major designs
are the Fassel and Greenfield torches. The end of this torch is placed inside an
induction coil supplied with a radio-frequency electric current. A flow of argon gas
(usually 14 to 18 liters per minute) is introduced between the two outermost tubes
of the torch and an electrical spark is applied for a short time to introduce free
electrons into the gas stream.
 These electrons interact with the radio-frequency magnetic field of the induction coil
and are accelerated first in one direction, then the other, as the field changes at
high frequency (usually 27.12 MHz or 40 MHz).
 The accelerated electrons collide with argon atoms, and sometimes a collision
causes an argon atom to part with one of its electrons. The released electron is in
turn accelerated by the rapidly changing magnetic field. The process continues until
the rate of release of new electrons in collisions is balanced by the rate of
recombination of electrons with argon ions (atoms that have lost an electron).
 This produces a ‘fireball’ that consists mostly of argon atoms with a rather small
fraction of free electrons and argon ions.
 INSTRUMENTATION
 An inductively coupled plasma (ICP) or transformer coupled plasma (TCP) [1] is a
type of plasma source in which the energy is supplied by electric currents which are
produced by electromagnetic induction, that is, by time-varying magnetic fields
Sampler: A nebulizer converts the sample to an aerosol that is introduced into the
excitation area of the plasma.
Source: The plasma jet source is made of three electrodes formed like a tripod. In each
arm there is a graphite anode and at the inverted base, a tungsten cathode is located. A
high-velocity inert gas, usually ICP argon, produces a high temperature plasma and
separates the excitation region from the analytical observation zone. The excitation area
is situated in the crook of the tripod and it has a temperature of 6,000 K. To increase the
current density and thus the plasma temperature it is necessary to squeeze the plasma in
order to decrease the current cross section. This is accomplished by cooling the edges of
the plasma with a high-velocity inert gas.
Analyzer: The analyzer is either a mono- or polychromator.
Detector: A photomultiplier converts radiant energy to measurable signals out.
APPLICATIONS OF ICP-MS
 Trace elemental analysis of water, soil, food samples etc.
 Determination Metallic poisons in viscera samples.
 Elemental analysis in biological fluids(blood, urine, etc.)
 Pharmaceutical analysis-traces of catalyst used, traces of poison metals(Cd,Pb,etc).
 Qualitative and quantitative analysis of metals and non-metals.
 Environmental analysis: trace metals and other elements in water, soils, plants,
composts, etc.
 Forensic examination: gunshot residue analysis, toxicological examination.

X-RAY SPECTROSCOPY
 X-rays were discovered by W.C. Rontgen in 1895. X-rays are a form of
electromagnetic radiation, wavelength ranging from 0.01nm to 10 nm. And energy
100 eV to 100KeV
 X-rays wavelength are shorter than those of UV rays and typically longer than those
of gamma rays. A technique used to determine the elements that are present and
their abundance in the sample. Also give bond length, angle.
GENERATION/PRODUCTION OF X-RAY (X-Ray Tube)
 X-rays are produced by accelerating electrons with a high voltage (50 kV to 80 kV)
and allowing with a high voltage and allowing them to collide with a metal target.
Features & Functioning of XRT:
 Composed of evacuated tube possessing cathode (tungsten filament) at one end &
anode (metal target) at another end.
 Passage of current through tube causes tungsten filament to glow & emits electron.
 Among the two electrodes large voltage difference is applied, causing electrons to
move at high velocity from filament and strike to anode.
 PRODUCTION OF X-RAYS
PRODUCTION OF X-RAYS
 There are three common mechanisms for the production of X-rays: the acceleration
of a charged particle, atomic transitions between discrete energy levels, and the
radioactive decay of some atomic nuclei. Each mechanism leads to a
characteristic spectrum of X-ray radiation.
 In the theory of classical electromagnetism, accelerating electric charges emit
electromagnetic waves. In the most common terrestrial source of X-rays, the X-ray
tube, a beam of high-energy electrons impinges on a solid target. As the fast-moving
electrons in the beam interact with the electrons and nuclei of the target atoms,
they are repeatedly deflected and slowed.
 During this abrupt deceleration, the beam electrons emit bremsstrahlung (German:
“braking radiation”)—a continuous spectrum of electromagnetic radiation with a
peak intensity in the X-ray region. Most of the energy radiated in an X-ray tube is
contained in this continuous spectrum. Far more powerful (and far larger) sources
of a continuum of X-rays are synchrotron particle accelerators and storage rings. In
a synchrotron, charged particles (usually electrons or positrons) are accelerated to
very high energies (typically billions of electron volts) and then confined to a closed
orbit by strong magnets.
 When the charged particles are deflected by the magnetic fields (and hence
accelerated via the change in their direction of motion), they emit so-
called synchrotron radiation—a continuum whose intensity and frequency
distribution are determined by the strength of the magnetic fields and the energy of
the circulating particles. Specially designed synchrotron light sources are
used worldwide for X-ray studies of materials.
 In an X-ray tube, in addition to the continuous spectrum of radiation emitted by the
decelerating electrons, there is also a spectrum of discrete X-ray emission lines that
is characteristic of the target material. This “characteristic radiation” results from
the excitation of the target atoms by collisions with the fast-moving electrons. Most
commonly, a collision first causes a tightly bound inner-shell electron to be ejected
from the atom; a loosely bound outer-shell electron then falls into the inner shell to
fill the vacancy.
 In the process, a single photon is emitted by the atom with an energy equal to the
difference between the inner-shell and outer-shell vacancy states. This energy
difference usually corresponds to photon wavelengths in the X-ray region of the
spectrum. Characteristic X-ray radiation can also be produced from a target
material when it is exposed to a primary X-ray beam. In this case, the primary X-
ray photons initiate the sequence of electron transitions that result in
the emission of secondary X-ray photons.
  Due to high velocity impact of electrons on to the target, inner shell electrons of
metal gets dislodge, which causes the outer shell electrons to jump to a lower energy
shell to replace the dislodge electrons.
 These electronic transitions results in the generation of x-rays. The produced x-rays
are allowed to move through a window of x-ray tube.
 The penetrating power of x-rays depends on energy also, there are two types of x-
rays.
1. Hard X-rays: which have high frequency and have more energy.
2. Soft X-rays: which have less penetrating and have low energy.
PRINCIPLE OF X-RAY FLUORESCENCE SPECTROSCOPY
 X-ray fluorescence (XRF) is the emission of characteristic fluorescent X-rays from a
material that has been excited by bombarding with high – energy X-rays.
 The wavelength of fluorescence is characteristic of the element being excited,
measurement of this wavelength enable us to identify the fluorescing element.
 The intensity of the fluorescence depends on how much of that element is in x-ray
beam.
 By measuring these energies determining the radiation emitted by the sample. it is
possible to determine which elements are present in sample this step is called
qualitative analysis.
 By measuring the intensities of the emitted energies, it is possible to determine how
much of each element is present in the sample. This step is called as quantitative
analysis.

INSTRUMENTATION OF X-RAY FLUORESCENCE SPECTROSCOPY

1. X-ray Tube
2. Sample Stage(Sample Preparation)
3. Collimator
4. Source Filters
5. Detectors
 There are two main types of XRF spectroscopy.
 1. Energy dispersive XRF (ED-XRF)
2. Wavelength dispersive XRF (WD-XRF)
 The main difference is the way, these fluorescent X-rays are detected and analysed.

Collimators
 Collimators are usually circular or a slit and restrict the size or shape of the source
beam for exciting small areas in either EDXRF or WDXRF instruments.
Source Filters
 Filters perform one of two functions
Background Reduction
Improved Fluorescence

SAMPLE PREPRATION
Powders:
 Grinding (<400 mesh if possible) can minimize scatter affects due to particle size.
 Pressing (hydraulically or manually) compacts more of the sample into the analysis
area, and ensures uniform density and better reproducibility.
Solids:
 Orient surface patterns in same manner so as minimize scatter affects.
 Polishing surfaces will also minimize scatter affects.
 Flat samples are optimal for quantitative results.
Liquids:
 Samples should be fresh when analyzed and analyzed with short analysis time - if
sample is evaporative.
 Sample should not stratify during analysis.
 Sample should not contain precipitants/solids, analysis could show settling trends
with time.
Advantages of XRF Analysis :-
 Rapid analysis
 Nondestructive analysis
 No spectrum is affected by chemical bonding
 Easily analysis of the element among the same family elements
 High accurate analysis
 Easy qualitative analysis
 Easy sample preparation
 Elemental carbon and sulfur can also be analyzed .
APPLICATIONS:
 Research in igneous, sedimentary, and metamorphic petrology
 Soil surveys
 Mining (e.g., measuring the grade of ore)
 Cement production
 Ceramic and glass manufacturing
 Metallurgy (e.g., quality control)
 Environmental studies (e.g., analyses of particulate matter on air filters)
XRF APPLICATION
 During the last decades, the development in X-ray detectors has established the
XRF method as a powerful technique in a number application fields, including:
 Ecology and environmental management: measurement of heavy metals in soils,
sediments, water and aerosols
 Geology and mineralogy: qualitative and quantitative analysis of soils, minerals,
rocks etc.
 Metallurgy and chemical industry: quality control of raw materials, production
processes and final products
 Paint industry: analysis of lead-based paints
 Jewelry: measurement of precious metals concentrations
 Fuel industry: monitoring the amount of contaminants in fuels
 Food chemistry: determination of toxic metals in foodstuffs
 Agriculture: trace metals analysis in soils and agricultural products
 Archaeology
 Art Sciences: study of paintings, sculptures etc.
X-RAY DIFFREACTION SPECTROSCOPY
PRINCIPLE:
 The first kind of scatter process to be recognized was discovered by Max von Laue
who was awarded the Nobel prize for physics in 1914 "for his discovery of the
diffraction of X-rays by crystals".
 X-ray diffraction (XRD) is a technique used to analyze the atomic and molecular
structure of a crystal, in which the crystalline atoms cause a beam of incident X-
rays to diffract into many specific directions.
 The atomic planes of crystal cause an incident beam of X-rays to interfere with one
another as they leave the crystal. The phenomenon is called X-ray diffraction.
  In X-ray diffraction, when X-rays, preferably of higher energy, hit the atoms that are
arranged in a material, they undergo two types of scattering: elastic or inelastic.
 In elastic scattering, the scattered rays have the same energy as the incident
electron while in inelastic scattering, the energy of the scattered rays is not equal to
the energy of the incident rays. Due to this, there is a constructive or destructive
interference based on the crystal orientation of the sample.
Bragg’s Law:
 Bragg’s law was used to explain the interference pattern of X-rays scattered by
crystals.
 Bragg’s Law was introduced by Sir W. H. Bragg and his son Sir W. L. Bragg.
 The law correlates the X – ray wavelength λ, interplanar spacing d, and reflection
angle θ.
 According to the law, when the x-ray is incident on to a crystal surface, its angle of
incidence will reflect back with a same angle of scattering.

 The constructive interference follows the Bragg law, which takes into account the
angle between the radiation and the crystal planes, the wavelength of the radiation,
and the spacing between the crystal planes. Through the Bragg equation, it is
possible to obtain information regarding the crystallinity of the sample.
 Typically, the analysis takes place either by fixing or varying the angle between the
incident ray and the crystalline planes.
 Metals such as copper, molybdenum, or iron are commonly used to generate the X-
ray sources by subjecting them to a high-voltage electron beam under vacuum
conditions. The samples can either by analyzed in a powdered form or as a whole
crystal. Based on the type of sample.
 INSTRUMENTATION
 X-ray Tube: the source of X rays
 Incident-beam optics: condition the X-ray beam before it hits the sample
 The goniometer: the platform that holds and moves the sample, and detector.
 The sample & sample holder
 Receiving-side optics: condition the X-ray beam after it has encountered the
sample
 Detector: count the number of X rays scattered by the sample

DETECTORS
 X-rays can be detected using two types of detectors:
1. Photographic Detectors
2. Counter Detectors
A. Geiger Muller Tube Counter
B. Scintillator Counter
Photographic Detectors
 To record the position and intensity of x-ray beam a plane or cylindrical film is used.
Cylindrical films are developed by exposing the detectors to X-rays. The film after
exposing to x-ray is developed.
 The extent of blackening of developed film is expressed as density.
 Density is the direct measurement of X-ray energy which causes blackening of
photographic film. The blackening of the developed film is expressed in terms of
density units D given by
D = log I₀/I,
I₀-incident intensity
I - Transmitted intensity
D - Total energy that causes blackening of the film D is measured by densitometer.
Geiger Muller Tube Counter:
 It is composed of glass tube (19 mm dia).
 The tube is comprised of a half metal cylinder of about 4 inches length, made up of
copper.
 Along the axis of cylinder, a thin metal wire of tungsten is tied.
 The cylinder & wire are connected to an electrical voltage source.
 The tube is filled with gas, usually Argon at a low pressure.
 A voltage is set up between the cathode and anode.

Scintillation Detector:
 Scintillation detector consists of scintillator and a device, such as PMT
(Photomultiplier tube) that converts the light into an electrical signal.
 Scintillator is a general term for substances that emit fluorescence when exposed to
radiation such as X-rays and γ-rays.
 When radiation collides with this substance (cerium-activated lithium or boron
silicates.), it absorbs its energy and internal electrons move from the ground state
(stable state) to the excited state (agitated state). When this electron returns to the
original stable state, it releases its energy in the form of light emission and this
phenomenon is called scintillation.
 The incident radiation can be measured quantitatively by photo-electrically
converting / amplifying the emitted fluorescence with a photomultiplier tube (PMT).
It is one of the common methods to measure invisible radiation.
APPLICATIONS OF XRD
 XRD is a non-destructive technique
 To identify crystalline phases and orientation
 To determine structural properties: strain, grain size, preferred orientation, order-
disorder transformation, thermal expansion
 To measure thickness of thin films and multilayers
 To determine atomic arrangement
 Biological macromolecular crystallography
FORENSIC APPLICATIONS READ FROM PPT SLIDES

NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

INTRODUCTION
 Nuclear magnetic resonance (NMR) spectroscopy is the study of molecules by
recording the interaction of radiofrequency (Rf) electromagnetic radiations with the
nuclei of molecules placed in a strong magnetic field.
 Zeeman first observed the strange behaviour of certain nuclei when subjected to a
strong magnetic field at the end of the nineteenth century, but the practical use of
the so-called “Zeeman effect” was only made in the 1950s when NMR spectrometers
became commercially available.
 Nuclear magnetic resonance (NMR) spectroscopy is a widely used analytical
technique for organic compounds. It is a spectroscopy technique which is based on
the absorption of electromagnetic radiation in the radio frequency region 4 to 900
MHz by nuclei of the atoms.
 NMR is based on the fact that the nucleus of each hydrogen atom in an organic
molecule behaves like a tiny magnet. NMR is the most valuable spectroscopic
technique used for structure determination.
Basis of NMR Spectroscopy
 Nuclear Magnetic Resonance (NMR) was first detected experimentally at the end of
1945, nearly concurrently with the work groups Felix Bloch, Stanford University
and Edward Purcell, Harvard University. The first NMR spectrum was first
published in the same issue of the Physical Review in January 1946. Bloch and
Purcell were jointly awarded the 1952 Nobel Prize in Physics for their research of
Nuclear Magnetic Resonance Spectroscopy.
 Nuclear magnetic resonance (NMR) spectroscopy is a crucial analytical tool for
organic chemists. The research in the organic lab has been significantly improved
with the aid of the NMR. Not only can it provide information on the structure of the
molecule, it can also determine the content and purity of the sample. Proton (1H)
NMR is one of the most widely used NMR methods by organic chemists. The protons
present in the molecule will behave differently depending on the surrounding
chemical environment, making it possible to elucidate their structure.
 More advanced NMR techniques are used in biological chemistry to study protein
structure. Spectroscopy determines the physical and chemical properties of atoms
or the molecules in which they are contained and provide detailed information about
the structure, dynamics, reaction state, and chemical environment of molecules. It
is used to study a wide variety of nuclei: – 1H, 13C.
  Nuclear magnetic resonance was first described and measured in molecular beams
by Isidor Rabi in 1938. Rabi was awarded the Nobel prize in physics for this work.
TYPES
 Two common types of NMR spectroscopy are used to characterize organic structure:
1. 1H NMR:- Used to determine the type and number of H atoms in a molecule.
2. 13C NMR:- Used to determine the type of carbon atoms in the molecule
PROTON OF NMR
 The most common of NMR is based on the hydrogen -1 (1H) nucleus, or 13 (13C)
 It can give information about the structure of any molecule containing hydrogen
atoms and carbon atoms.
PRINCIPLE OF NMR
 The principle is based on the- spinning of nucleus and generating a magnetic field.
Without external magnetic – field nuclear spin are random in direction.
 With nuclei align themselves either with or against field of external magnetic field.
If an external magnetic field is applied, an energy transfer (ΔE) is possible between
ground state to excited state. When the spin returns to its ground state level, the
absorbed radiofrequency energy is emitted at the same frequency level. The emitted
radiofrequency signal that give the NMR spectrum of the concerned nucleus.
 Many nuclei have spin, and all nuclei are electrically charged, according to the
NMR principle. An energy transfer from the base energy to a higher energy level is
achievable when an external magnetic field is supplied.
 All nuclei are electrically charged and many have spin.
 Transfer of energy is possible from base energy to higher energy levels when an
external magnetic field is applied.
 The transfer of energy occurs at a wavelength that coincides with the radio
frequency.
 Also, energy is emitted at the same frequency when the spin comes back to its
base level.
 Therefore, by measuring the signal which matches this transfer the processing
of the NMR spectrum for the concerned nucleus is yield.

 In NMR we put the sample to be analyzed in a magnetic field.

 The hydrogen nuclei (protons) either line up with the field or, by spinning in the
opposite direction, line up against it.
  Some nuclei experience this phenomenon, and others do not, dependent upon
whether they possess a property called spin.
 In a magnetic field, there are now two energy states for a proton:
1. A lower energy state with the nucleus aligned in the same direction as magnetic
field.
2. A higher energy state in which the nucleus aligned against to magnetic field.

INSTRUMENTATION
1. Sample holder
2. Permanent magnet
3. Magnetic coils
4. Sweep generator
5. Radio frequency transmitter
6. Radio frequency receiver
[Link] out systems
NMR Spectroscopy Working
 Place the sample in a magnetic field.
 Excite the nuclei sample into nuclear magnetic resonance with the help of radio
waves to produce NMR signals.
 These NMR signals are detected with sensitive radio receivers.
 The resonance frequency of an atom in a molecule is changed by the intramolecular
magnetic field surrounding it.
 This gives details of a molecule’s individual functional groups and its electronic
structure.
 Nuclear magnetic resonance spectroscopy is a conclusive method of identifying
monomolecular organic compounds.
 This method provides details of the reaction state, structure, chemical environment
and dynamics of a molecule.
[Link] holder: - Glass tube with 8.5 cm long,0.3 cm in diameter.
[Link] magnet: - It provides homogeneous magnetic field at 60-100 MHZ.
[Link] coils: - These coils induce magnetic field when current flows through them.
[Link] generator: - To produce the equal amount of magnetic field pass through the
sample.
[Link] frequency transmitter: - A radio transmitter coil that produces a short powerful
pulse of radio waves.
[Link] receiver: - A radio receiver coil that detects Receiver radio frequencies
emitted from the sample matrix.
[Link] system: - A computer that analyses and record the data
 NMR spectroscopy works by varying the machine’s emitted frequency over a small
range while the sample is inside a constant magnetic field.
 Most of the magnets used in NMR machines to create the magnetic field range from
6 to 24 T.
 The sample is placed within the magnet and surrounded by superconducting coils,
and is then subjected to a frequency from the radio wave source. A detector then
interprets the results and sends it to the main console.
FORENSIC APPLICATIONS OF NMR
 Drug analysis NMR spectroscopy has been used by scientists for drug analysis for
many years.
 Various pharmaceutical industries use this spectroscopic technique to identify
drugs and their metabolites.
 The applications of 1H NMR and 13C NMR spectroscopy for the analysis of different
drugs have been very well reviewed in the past.
 Over the years, forensic scientists and investigators have started using NMR to
identify drugs in different samples or evidence collected from the crime scene.
 NMR spectroscopy is a Spectroscopy technique used by chemists and biochemists
to investigate the properties of organic molecules, although it is applicable to any
kind of sample that contains nuclei possessing spin.
 For example, the NMR can quantitatively analyze mixtures containing known
compounds. NMR can either be used to match against spectral libraries or to infer
the basic structure directly for unknown compounds.
 Once the basic structure is known, NMR can be used to determine molecular
conformation in solutions as well as in studying physical properties at the molecular
level such as conformational exchange, phase changes, solubility, and diffusion.
 SEPARATION AND DETECTION TECHNIQUES
 Chromatography, literally ‘’colour writing’’, was first employed by Russian-Italian
scientist Mikhail Tsvet in 1906, primarily for the separation of plant pigments such
as chlorophyll, carotenes and xanthophylls.
 The word chromatography is derived from two Greek words
Chroma – colour
Graphos – writing
Definition: A method of separating a mixture of components into individual components
through equilibrium distribution between two phases.
Principle of Chromatography:
 it is works on the principle of Partition coefficient, is defined as the molar
concentration of analyte in the stationary phase divided by the molar concentration
of the analyte in the mobile phase.
 The technique of chromatography is based on the differences in the rate at which
the components of mixture moving through a porous medium (stationary phase)
under the influence of some solvent or gas (moving/mobile phase).

1. Adsorption Chromatography:
 Adsorption chromatography is probably one of the oldest types of chromatography
around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface
of a stationary phase.
 The equilibration between the mobile and stationary phase accounts for the
separation of different solutes.
 Ex: Column Chromatography, TLC, HPTLC, Gas Solid Chromatography.
2. Partition Chromatography:
 This form of chromatography is based on a thin film formed on the surface of a solid
support by a liquid stationary phase. Solute equilibrates between the mobile phase
and the stationary liquid.
 Separation of components of a sample mixture occurs because of partition.
Stationary phase is coated with liquid which is immiscible in mobile phase.
 The component of sample mixture appear separated because of differences in their
partition coefficient.
Ex: Liquid-Liquid Chromatography, Gas Liquid Chromatography.

3. Ion Exchange Chromatography:

 Ion exchange chromatography is a process that allows the separation of ions and
polar molecules based on their affinity to the ion exchanger.
 It can be used for almost any kind of charged molecules including large protein,
small nucleotide and amino acids etc. It is often used in protein purification, water
analysis, and quality control.
 Ion chromatography is used to separate organic or inorganic charged substances.
The stationary phases used are based on typical ion exchange resins.
4. Size Exclusion Chromatography:
 It is a chromatographic method in which molecules in a solution are separated by
their size. It is usually applied to large molecules or macromolecular complexes such
as protein and industrial polymers.
 A mixture of molecules dissolved in liquid (mobile phase) is applied to
chromatography column which contains a solid support in the form of beads
(stationary phase).
 The mass of beads within the column is often referred to as the column bed. The
beads act as traps or sieves and function to filter small molecules which become
temporarily trapped within the pores.
 Large molecules are excluded from the beads. Large sample molecules cannot or
can only partially penetrate the pores, whereas smaller molecules can access most
or all pores.
 Thus large molecules elute first, smaller molecules elute later, while molecules that
can access all the pores elute last from the column.

Thin Layer Chromatography

 The discovery of thin-layer chromatography (TLC) is attributed to Izmailov and
Shraiber, who, in 1938, separated plant extracts by spotting them onto adsorbent
layers produced by coating microscope slides with a slurry of the adsorbent with
water and allowing to dry.
 Stationary phase— A Thin layer of coating (usually alumina or silica gel) on a sheet
of Glass/Aluminium foil /plastic
 Mobile phase—a liquid solvent
 Components of TLC: TLC Plates, TLC Chamber, Mobile Phase, Filter paper,
Detecting or visualizing agents (Spraying Reagent)
 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
 High performance liquid chromatography or commonly known as HPLC is an
analytical technique used to separate, identify or quantify each component in a
mixture.
 The mixture is separated using the basic principle of
column Chromatography and then identified and quantified by spectroscopy.
 In the 1960s the column chromatography with its low-pressure suitable glass
columns was further developed to the HPLC with its high-pressure adapted metal
columns.
 it is especially suitable for compounds which are not easily volatalised, thermally
unstable and have high molecular weights.
PRINCIPLE:
 The purification takes place in a separation column between a stationary and a
mobile phase.
 The stationary phase is a granular material with very small porous particles in a
separation column.
 The mobile phase, on the other hand, is a solvent or solvent mixture which is forced
at high pressure through the separation column.
 Via a valve with a connected sample loop, i.e. a small tube or a capillary made of
stainless steel, the sample is injected into the mobile phase flow from the pump to
the separation column using a syringe.
 Subsequently, the individual components of the sample migrate through the column
at different rates because they are retained to a varying degree by interactions with
the stationary phase.
 After leaving the column, the individual substances are detected by a suitable
detector and passed on as a signal to the HPLC software on the computer.
 At the end of this operation/run, a chromatogram in the HPLC software on the
computer is obtained.
 The chromatogram allows the identification and quantification of the different
substances.

INSTRUMENTATION
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
 The Pump
 Injector
 Column
 Detector
 Recorder
The Pump:
• The development of HPLC led to the development of the pump system.
• The pump is positioned in the most upper stream of the liquid chromatography
system and generates a flow of eluent from the solvent reservoir into the system.
• High-pressure generation is a “standard” requirement of pumps besides which, it
should also to be able to provide a consistent pressure at any condition and a
controllable and reproducible flow rate.
Injector:
• An injector is placed next to the pump.
• The simplest method is to use a syringe, and the sample is introduced to the column.
• The most widely used injection method is based on sampling loops.
• The use of the autosampler (auto-injector) system is also widely used that allows
repeated injections in a set scheduled-timing.
Column:
• The separation is performed inside the column.
• The recent columns are often prepared in a stainless steel housing, instead of glass
columns.
• The packing material generally used is silica or polymer gels.
• Most column housing is made of stainless steel since stainless is tolerant towards
a large variety of solvents.

Detector: (DAD)
• Separation of analytes is performed inside the column, whereas a detector is used
to observe the obtained separation.
• The composition of the eluent is consistent when no analyte is present. While the
presence of analyte changes the composition of the eluent. What detector does is to
measure these differences.
• This difference is monitored as a form of an electronic signal. There are different
types of detectors available.
Recorder:
• The change in eluent detected by a detector is in the form of an electronic signal,
and thus it is still not visible to our eyes.
• In older days, the pen (paper)-chart recorder was popularly used. Nowadays, a
computer-based data processor is more common.
• There are various types of data processors; from a simple system consisting of the
in-built printer and word processor while those with software that are specifically
designed for an LC system which not only data acquisition but features like peak-
fitting, baseline correction, automatic concentration calculation, etc.
Types of High-Performance Liquid Chromatography (HPLC)
• Normal phase
• Reverse phase
• Ion exchange
• Size exclusion
Applications of HPLC
The information that can be obtained by HPLC includes resolution, identification and
quantification of a compound. It also aids in chemical separation and purification. The
other applications of HPLC include :
Applications in Forensics
 Quantification of drugs in biological samples.
 Identification of steroids in blood, urine etc.
 Forensic analysis of textile dyes.
 Determination of cocaine and other drugs of abuse in blood, urine etc.
Environmental Applications
 Detection of phenolic compounds in drinking water.
 Bio-monitoring of pollutants.
Pharmaceutical Applications
 To control drug stability.
 Tablet dissolution study of pharmaceutical dosages form.
 Pharmaceutical quality control.
Food and Flavour
• Measurement of Quality of soft drinks and water.
• Sugar analysis in fruit juices.
• Analysis of polycyclic compounds in vegetables.
• Preservative analysis.
Applications in Clinical Tests
• Urine analysis, antibiotics analysis in blood.
• Analysis of bilirubin, biliverdin in hepatic disorders.
• Detection of endogenous Neuropeptides in extracellular fluid of brain etc.
 INTRODUCTION TO GAS CHROMATOGRAPHY
GC
GC-MS/GC-MS-MS
GC-HSS
LC-MS/LC-MS-MS
 The father of modern gas chromatography is Nobel prize winner John Porter Martin,
who also developed the first liquid gas chromatography.
 It is a process of separating components from the given crude drug by using a
gaseous mobile phase.
 It involves a sample being vaporized and injected onto the head of the
chromatographic column.
 The sample is transported through the column by the flow of inert, gaseous mobile
phase.
 The column itself contains a liquid stationary phase which is a adsorbed onto the
surface of an inert solid.
1. Gas – Solid Chromatography:
Mobile Phase – Gas
Stationary Phase – Solid
2. Gas – Liquid Chromatography:
Mobile Phase – Gas
Stationary Phase – Liquid
PRINCIPLE OF GAS CHROMATOGRAPHY
 Gas Chromatography is a technique in which the Components of a mixture in the
gaseous state are separated by passing the sample through a column containing
(stationary phase) under the influence of gaseous mobile phase.
 It works on the principle of “Partition”.
 The component which is more soluble in stationary phase travel slower and eluted
later. The component which is less soluble in stationary phase travels faster and
eluted out first.

INSTRUMENTATION
COMPONENTS OF GC
1. Carrier Gas – He(Common), N2, H2, Argon.
2. Sample Injection Port – Micro Syringe.
3. Columns
4. Oven
5. Detectors – FID, TCD, ECD, NPD.

GC-CARRIER GAS:
 Carrier gas is An inert gas, which is used to sweep a mixture to be separated through
a gas chromatograph, (helium, hydrogen, or nitrogen).
 Push the sample through the gas chromatograph column.
 Clean out the gas chromatograph column after sample analysis.
 The Flow mode has four options for the carrier gas control:
1. Constant flow
2. Constant pressure
 [Link] flow
[Link] pressure
 The criteria for selecting the carrier gas
 It should be chemically inert
 It should be non-toxic.
 It should be non-reactive.
 It should be non-flammable.
 It should be compatible with the column detector.
 It should give good separation efficiency.
 It should be pure (moisture and oxygen free).
 It should give best possible result.
 It should be suitable for the sample to be analyzed and for the detector.
 Nitrogen and Argon are preferred for packed columns
 Flow rate : 25- 150 mL/min for packed columns
 Hydrogen and Helium are preferred for open tubular columns
 Flow rate : 2-25 mL/min for open tubular columns
 But due to cost factor, Nitrogen is the choice for packed column and Helium is
the choice for open tubular columns due to the inflammable nature of Hydrogen.
GC- SAMPLE INJECTION
SAMPLE INJECTION:
 Sampling unit or injection port is attached to the column head.
 The aim of the injection is to introduce the sample as a sharp band into the carrier
gas stream.
 The samples for GC can be either liquids, solids, or gases.
 Solids and in most cases liquids are usually injected using a micro syringe (0.1-100
µL) as dilute solutions in a volatile solvent.
 Gaseous samples may be introduced by use of a gas tight hypodermic needle of 0.5
– 10 ml capacity.
 The solvent is un retained and passes straight through the column to give a solvent
peak.
 Since, the sample should be in vaporized state, the injection port is provided with
an oven that helps to maintain its temperature at about 20-50 oC above the boiling
point of the sample..
Injection of Samples into capillary columns:
1. Split Injection:
 Only small portion (may be 1-10%) of the sample moves into the column, and the
rest is sent to waste. This is used when the analytes are in high concentration.
2. Split less Injection: (where the split vent is closed)
 Attempts to transfer all of the sample to the column and is used for trace analysis.

Components of GC- Column:

• Column is the heart of the GC System
• The column is where the chromatographic separation of the sample occurs.
• Several types of columns are available for different chromatographic applications
• It is coated with a stationary phase which greatly influences the separation of the
compounds.
• Stationary phase : Solid resin packed in a column/ Liquid supported by an Inactive
solid over which a mixture passes.
• Each component of the mixture differs in the way it adheres to this phase and
therefore travels along it at a unique rate.
DETECTORS
 The eluted solute particles along with the carrier gas exit from the column and enter
the detector.
 The detector then produces electrical signals proportional to the concentration of
the components of solute.
 The signals are amplified and recorded as peaks at interval on the chromatograph.
1. Flame Ionization detector (FID)
2. Thermal Conductivity Detector (TCD)
3. Electron Capture Detector (ECP)
4. Flame Photometric detector (FPD)
5. Nitrogen and Phosphorous Detector (NPD)
FLAME IONIZATION DETECTOR
 This employs hydrogen flame that is maintained in a small cylindrical jet made up
of platinum or quartz.
 Effluent from the column with helium or nitrogen as carrier gas are fed into the
hydrogen flame, gets ignited and undergoes pyrolysis to produce ions.
 For detection of these ions, two electrodes are used that provide a potential
difference.
 The ions produced are repelled by the positive electrode which hit the collector
plate.
 The current produced in doing so is amplified and fed to an appropriate recorder.
GC-HEAD SPACE APPLICATIONS
• Blood alcohol analysis.
• Analysis of alcohol content in sanitizers
• Analysis of Fire Debris for inflammable hydrocarbons
• Determination of volatile hydrocarbons in waste water.
• Determination of residual volatile solvents in pharmaceutical.
• Monomers in polymers determination ex: styrene
• Determination of out-gassing solvents from packages.
• Flavour profiles in drinking beverages or foods.
FORENSIC APPLICATIONS:
• Environmental monitoring
• Medicine and Pharmaceutical Applications
• Security and chemical warfare Agent Detection
• Forensic applications
• Cases of Drug Abuse,
• Arson
• Blood alcohol,
• In Forensic Toxicology- poison & pesticide analysis in biological materials such as
viscera , blood etc.
• Qualitative and Quantitative analysis
• In determining the levels of metabolites in body fluids like blood, urine etc.
• Analysis of foods like carbohydrates, proteins, lipids, vitamins, steroids, drug and
pesticides residues, trace elements.
• Pollutants like formaldehyde, carbon monoxides, benzene, etc.
• Separation and identification of volatile materials, plastic, natural and synthetic
polymers, paints, and microbiological samples.
DISADVANTAGES OF GC:
 GC is suitable only for volatile &thermally stable compounds
 GC is not suitable for Thermally labile compounds.
 GC do not give a specific identification of the compound
 By combining GC with Mass Spectroscopy constituents of the mixtures can be
specifically identified.
GC-MS:
• Chromatography produces pure fractions of chemical components in a mixture.
• Spectroscopy produces selective information for identification using standards or
library spectra
 • By combining GC with Mass Spectroscopy constituents of the mixtures can be
specifically identified.
• Mass Spectrometry is an analytical method to measure the molecular or atomic
weight of samples.
• The concept of mass spectrometry was first put forth by Sir J.J. Thomson, English
Physicist who discovered the electron in 1887.
• He got Nobel prize in Physics 1906.
What does a mass spectrometer do?
 Mass spectrometer is easy technique to give you Molecular weight (from molecular
ion(M+))
 You can get molecular formula (Elements present)
 Nearly all elements in the periodic table can be determined by mass spectrometry.
 It can give information about Chemical Structure as well.
LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LC-MS)
 Liquid chromatography (LC) is a separation technique, first demonstrated in the
early 1900s by Russian botanist, Mikhail Tswett. LC separates the components of a
sample based on the differences in their affinity or retention strength for the
stationary phase and mobile phase.
 The Liquid Chromatography-Mass Spectrometry (LC-MS) is hyphenated analytical
technique which is combination of Liquid Chromatography (LC) and Mass
Spectrometry (MS).
 HPLC (LC) separates the components of mixtures by passing through
chromatographic column. Generally, the separated components cannot be positively
identified LC alone.
 Mass Spectrometry is also used for identification of unknown compounds, known
compounds and to elucidate the structure.
 pH & BUFFERS & PHYSIOLOGICAL SOLUTIONS
pH DEFINITION:
 pH is a measure of how acidic/basic water is.
 The range goes from 0 - 14, with 7 being neutral. pH of less than 7 indicate acidity,
whereas a pH of greater than 7 indicates a base. (Or) pH is really a measure of the
relative amount of free hydrogen and hydroxyl ions in the water.
SORENSEN’S pH SCALE:
  The concept of pH scale was first introduced by Danish chemist Soren Peder
Lauritz Sorensen (S.P.L. Sorensen), in the year 1909.
 The scale was later revised to the modern pH in the year 1924.
 Sorenson defined pH as the logarithm of the reciprocal of hydrogen ion
concentration (Or, it can be rearranged as)
 pH is a term used to specify the acidity or basicity of an solution. A pH scale helps
in measuring how acidic or basic a substance.
 Thus, pH may be defined as negative logarithm of hydrogen ion concentration.

Sorensen’s pH Scale
 Based on the pH values and different concentrations of H+ ions, a scale has been
devised and named after Sorensen.
 The scale starts with zero pH, i.e, hydrogen ion concentration is 10º. It means the
solution is strongly acidic.
 At the other end of the scale, pH is 14. i.e, hydrogen ion concentration is 10 -14. It
means the solution is strongly alkaline.
 The central point pH in the scale is 7.0, because [H+] is equal to [OH+], i.e., hydrogen
ion concentration is 10-7
 Solutions with a pH less than 7 are acidic and solutions with a pH greater than 7
are basic.
 Pure water neutral, being neither an acid nor a base.
 pH Applications: Enhancing solubility, increasing stability, improving purity,
Optimizing biological activity and storage of products.
ACIDS
 An acid is any substance that dissociates (ionizes) in solution and releases hydrogen
ions (H+).
 Acids have the following characteristics:
• Taste sour
• Turns litmus indicators red
• React with bases to form salts
Examples of acids in body includes: Hydrochloric acid, lactic, phosphoric, carbonic, citric
and carboxylic acids.
BASES
 A base is any substance that picks up or accepts H+ to form hydroxide ions (OH-)
in water solutions.
 Base or alkaline solutions have the following characteristics:
• Bitter taste
• Slippery to the touch
• Turns litmus indicators blue
• React with acids to form water
Examples of bases in the body include sodium and calcium hydroxide and aqueous
solutions of ammonia that form ammonium hydroxide.
LITMUS PAPER
  To test whether the solution is acidic or alkaline.

BUFFER
 “Buffers” are compounds or mixtures of compounds that by their presence in the
solution resist changes in the pH upon the addition of small quantities of acid or
alkali.”
 For an acid- buffer solution, it consists of a weak acid and its conjugate base.
 For a basic- buffer solution, it consists of a weak base and its conjugate acid.
 Typically a mixture of a weak acid and a salt of its conjugate base or weak base and
a salt of its conjugate acid.
TYPES OF BUFFER: Generally buffers are of two types :-
1. Acidic buffers
2 . Basic buffers
1. ACIDIC BUFFERS An acidic buffer is a combination of weak acid and its salt with a
strong base. i.e. Weak acid and salt with strong base (conjugate base).
 Example –
• CH3COOH / CH3COONa
• H2CO3 / NaHCO3
• H2PO4 / NaHPO4
• HCOOH / HCOONa
2. BASIC BUFFERS A basic buffer is a combination of weak base and its salt with a strong
acid. i.e. Weak base and salt with strong acid (conjugate acid).
 Example:
• NH4OH / NH4Cl
• NH3 / NH4Cl
• NH3 / (NH4)2CO3

APPLICATION OF BUFFERS:
 To improve purity
  To increase stability
 To enhance solubility
 To optimize biological activity
 To maintain constant pH
PHYSIOLOGICAL BUFFER SOLUTIONS
 Importance of Buffers in Physiological Systems: Processes that take place in living
organisms are called physiological processes.
 Like blood circulatory system, respiration etc.
 The internal pH of most living cells is close to 7.0.
 The pH of human blood is 7.4.
 A blood pH of below 7 or above 7.8 can cause death within minutes.
 So buffering of blood pH is very important to stabilize it around 7.4. pH plays
an important role in almost all biological processes.
 Small change in pH i.e. deceased or high pH can cause metabolic implications in
human body like acidosis and alkalosis.
 Where metabolism is involved there would be definitely a need of buffer as within
cells metabolism is associated with the release of protons (H+) i.e. decrease in pH or
uptake of protons (H+) i.e. increase in pH.
 Important buffers that are dominant in human body are:
1. Bicarbonate buffers
2. Phosphate buffers
3. Protein buffers
Bicarbonates buffers (Buffering in blood)
 Blood is a biological fluid in which Carbonic acid and Hydrogen carbonate buffer
system plays an important role in maintaining pH around In this buffer, carbonic
acid (H2CO3) act as a weak acid and hydrogen carbonate ion (HCO3-) act as
conjugate base of a weak acid. H2CO3 ↔ H+ + HCO3-
 When there is excessive amount of H+ in the blood it is consumed by HCO3- forming
carbonic acid that is a weak acid which does not alter the blood pH so much and
when there is excessive amount of OH- in the blood it is consumed by H2CO3 as it
will release the H+ ions upon excess amount of OH- in the blood forming H2O.
 Proportion of carbonic acid and hydrogen carbonate is also very much important in
blood.
 Carbonic acid concentration is controlled by respiration through lungs while
hydrogen carbonate concentration is controlled by urination through kidneys.
 Carbonic acid buffer system is a critical buffer for blood as in the absence of this
buffer system the pH may fall below this normal value within blood producing a
condition a condition called acidosis ( acidosis may be respiratory or metabolic
acidosis) or the pH may rise above normal level producing a condition known
as alkalosis (alkalosis may be respiratory or metabolic alkalosis).
Phosphate buffer (Buffering of internal cell fluids)
 The phosphate buffer system works in the internal fluid of all cells.
 This buffer system consists of dihydrogen phosphate ions (H2PO4-) as a weak acid
and hydrogen phosphate ions (HPO42-) as a conjugate base of weak acid.
 These two ions are in equilibrium with each other as indicated by the chemical
equation below.
H2PO4- ↔ H+ + HPO42-
 If additional hydrogen ions enter the cellular fluid, they are consumed in the
reaction with HPO42-, and the equilibrium shifts to the left.
  If additional hydroxide ions enter the cellular fluid, they react with H2PO4-,
producing HPO42-, and shifting the equilibrium to the right.
 In the absence of phosphate buffer from cell fluid, sharp changes in pH of cell fluids
may cause cell death or improper working of different proteins and cell organelles
present within the cell.
Protein buffer (Buffering in Cells and Tissues)
 Proteins are mainly composed of amino acids.
 These amino acids contain functional groups that act as weak acid and bases when
there are sharp changes in pH in order to stabilize the pH within the body cells.
 In short it can be said that proteins act as buffers themselves.
 Protein is a significant buffer the main buffering site for protein is cells and tissues
but even in blood it act as a buffer consuming hydrogen ions produced due to the
dissociation of the carbonic acid into hydrogen bicarbonate.
 To understand the proteins as a buffer we have to look into the structure of amino
acids which consists of
carboxyl group (COOH)
amino group (NH2)
CENTRIFUGATION
 English military engineer Benjamin Robins (1707–1751) invented a whirling arm
apparatus to determine drag.
 In 1864, Antonin Prandtl proposed the idea of a dairy centrifuge to separate cream
from milk.
 The idea was subsequently put into practice by his brother, Alexander Prandtl, who
made improvements to his brother's design, and exhibited a working butterfat
extraction machine in 1875
 A centrifuge is a device that uses centrifugal force to separate various components
from a fluid. This is done by spinning fluid at high speeds within a container.
 It can then separate fluids with different densities (e.g. Cream from milk, or liquids
and solids.
 It is a technique which involves the application of centrifugal force to separate
particles from a solution according to their size, shape, density and viscosity of the
medium and rotor speed.
 The centrifuge is widely employed in labs for getting biological compounds
separated from crude extract.
 In a centrifuge, the specimen is placed in a rotating device that rotates around an
axis (axis) that results in an intense force that is perpendicular towards the axis.
 There are various kinds of centrifuges that are used for the separation of various
molecules, however they all operate on the same principle of sedimentation.
PRINCIPLE OF CENTRIFUGATION
 The centrifuge involve the principle of sedimentation.
 The principle of the centrifugation is to separate the particles suspended in liquid
media under the influence of a centrifugal field.
 Sedimentation is a phenomenon where suspended material settles out of the fluids
by gravity. The suspended material can be particles such as clay or powder etc.
 In a solution, particles whose density is higher than that of the solvent sink
(sediment), and particles that are lighter than it floats to the top.
 The greater the difference in density, the faster they move. If there is no difference
in density (isopycnic conditions), the particles stay steady.
CENTRIFUGAL FORCE
 Centrifugal force is an outward fictitious force that is experienced by an object
moving in a circular path directed away from the center of rotation.
  The formula to calculate the centrifugal force is given below.
 If the velocity of the moving object is known, the centrifugal force can be calculated
by the formula:

Where, v is the velocity of the moving body, r is the distance of the moving body from the
center and m is the mass of the moving body.
 If the angular velocity of the moving object is known, the centrifugal force can be
calculated by the formula:

Where, ω is the angular velocity, r is the distance of the moving body from the center
and m is the mass of the moving body.

Relative Centrifugal Force (RCF)

 Relative centrifugal force is a measurement for the rate of strength in rotors that
are of different sizes and types.
 The force is that is exerted on the inside of the rotor by the force of the rotor’s
rotation.
 RCF is the force perpendicular to the surface applied to the sample, which is always
in relation to the gravity of the earth.
 The RCF of different centrifuges is a good tool for analysis of rotors.
 The formula used to calculate the force of centrifugal relative (RCF) could be written
as follows:

Where as,
RCF – The relative centrifugal field in units of ‘g’
RPM – The speed of rotation in revolution per minute
R – The radius of rotation in centimeters
CENTRIFUGATION PROCESS
 The centrifuge is an container inside which an amalgamation of liquid and solid or
two liquids are placed. Then, the container is turned at a very high speed.
 As the container rotates at high speed, the mixture is separated into its components
by the effect of centrifugal force upon their density.
SEDIMENTATION PRINCIPLE OF CENTRIFUGE
  In a solution, the particles with a density that is greater than that of the sink
(sediment) and those which are lighter than the sink, float up to the top.
 The more different density, the more quickly they travel. If there isn’t any distinction
between the intensity (isopyknic conditions) the particles remain in a steady state.
 To make the most of tiny variations in density, to distinguish different particles
within a solution gravity is replaced by the more potent “centrifugal force” that is
provided by centrifuge.
The forces works during the centrifugation
Two forces oppose the centrifugal force that acts on suspended particles:
 Buoyant force: force by which particles have to move away from the liquid medium
into which they settle.
 Frictional force: force created by particles when they move throughout the liquid.
Particles are able to move off the plane of motion in the field of a centrifugal force only
when the force of centrifugal forces exceeds the buoyant and frictional counteracting
forces, resulting in the sedimentation of particles in a steady rate.

TYPES OF CENTRIFUGATION TECHNIQUES

1. Preparative Centrifugation
 For separation of one type of materials from other.
2. Analytical Centrifugation
 For measurement of physical properties of macromolecules.
LOW-SPEED CENTRIFUGE
 Low-speed centrifuges are the most common centrifuges commonly used in labs to
perform the regular sorting of the particles.
 The centrifuges are operated at the maximum speed of 4000-5000 rpm.
 They are typically operated at ambient temperature since they don’t have control
mechanisms to regulate the speed or the temperature of the process.
 Fixed angle and swinging bucket types of rotors are used in these centrifuges.
 They are compact and easy to use centrifuges, which are great for the analysis of
blood samples as well as different biological sample.
 The centrifuge that operates at low speed follows the same principle that is used in
all other centrifuges, however the application is limited to removal between simpler
and more efficient solutions.
HIGH-SPEED CENTRIFUGES
 High-speed centrifuge as the name implies is the type of centrifuge which can
operate at speeds that are slightly higher.
 Speed of the centrifuge with high speed may vary between 15,000 and 30,000 rpm.
 The high-speed centrifuge is typically employed in laboratories that are more
sophisticated for biochemical applications and requires a fast speed of operation.
 High-speed centrifuges have an instrument for controlling both the temperature and
speed of the procedure that is crucial to analyze sensitive biological molecules.
 The high-speed centrifuges are equipped with different adapters to fit samples tubes
of different dimensions and volumes.
 The three types of rotors may be used to create the centrifugation process of these
centrifuges.
[Link] Angle rotor
[Link] bucket/Horizontal rotor
[Link] rotor
ULTRACENTRIFUGES
 Svedberg coined the term “ ultracentrifugation".
 Ultracentrifuges run at very high speeds which allow separation of smaller
molecules like proteins, ribosomes and even viruses.
  This is the highest advanced type of centrifuge which allows to separate molecules
which can’t be separated by other centrifuges.
 Refrigeration systems are found in these centrifuges to help control the heat that is
generated by the spinning.
 They could be up to 150,000 rpm.
 It can be used to aid in both analytical and preparation work.
 Ultracentrifuges can be used to separate molecules in large batches , and in a
continuous flow.
 Apart from separation, ultracentrifuges may also be employed for the analysis of
macromolecules’ characteristics, including the shape, size, and density.
MICROCENTRIFUGES
 Microcentrifuges can be employed to separate small volumes of samples that range
from 0.5 and 2 µl.
 Microcentrifuges usually operate at around 12,000-13,000 rpm.
 This is utilized for to separate molecularly organelles in cells like DNA, nuclei as for
extraction with phenol.
 Microcentrifuges also referred to as microfuge, utilize test tubes which are smaller
as compared to the traditional test tubes that are utilized in larger centrifuges.
 Certain microcentrifuges have adapters that allow making use of bigger tubes with
smaller ones.
 Microcentrifuges with temperature controls are available for the operation of
temperature-sensitive samples.
BENCHTOP CENTRIFUGE
 Benchtop centrifuges are compact centrifuge, which is widely used in research and
clinical laboratories.
 It is powered through an electric motor. the tubes rotate around an undetermined
axis, which results in force perpendicularly towards the tubes.
 Because they are small, they can be useful for smaller labs that have smaller space.
 A variety of centrifuges for benchtop use are available on the market for a variety of
uses.
 A benchtop centrifuge is equipped with an rotor that has racks for the tubes that
are used to test and a lid that covers the unit that is used to operate the centrifuge.
DENSITY GRADIENT CENTRIFUGATION
 Density gradient centrifugation refers to an approach to separation between
molecules in which the separation is determined by the concentration of
molecules as they move through a density gradient under the influence of a
centrifugal force.
Principle of Density gradient centrifugation
 Density gradient centrifugation relies on the idea that molecules settle under the
force of centrifugal forces until they find the same density in a medium similar to
their own.
 In this instance the medium has the density gradient is utilized that needs to reduce
density or increase density.
 The molecules that are more dense start to shift towards the bottom of the gradient
as they travel through the densities gradient. The molecules are then suspended at
a level at which the density of particles is greater than that of the medium. This way
molecules of different density are separated into various layers. They can be
recovered through various methods.
 This type of centrifugation is mainly used to purify viruses, ribosomes, membranes,
etc.
Steps of Density gradient centrifugation
1. A density gradient in the medium is produced by gently spreading the
concentrations with lower levels over more concentrated ones in the centrifuge tube.
2. The sample is placed over the gradient after which the tubes will be then placed
inside an ultracentrifuge.
3. The particles move along their gradients until they arrive at a point where their
density is equal to with the denseness of medium around them.
4. The fragments are then removed and separated, giving particles that are isolated.
Uses of Density gradient centrifugation
 Density gradient centrifugation may be used to purify huge quantities of
biomolecules.
 It could even be utilized to purify various viruses, which can aid in their future
research.
 This method can be utilized to separate particles as well as a method to determine
the densities of different particles.
 Examples of centrifugation using a density gradient
 This technique was utilized for the long-running experiment which demonstrated
that DNA was semi-conservative employing different nitrogen isotopes.
 Another instance is the use of this method for the removal of the microsomal fraction
in muscle homogenates and then the segregation of membrane vesicles that have
different density.
 There are 2 forms of density gradient centrifugation one is rate zonal centrifugation
and the second is isopycnic or sedimentation equilibrium centrifugation.
A) Rate zonal centrifugation:
B) Isopycnic or sedimentation equilibrium centrifugation
A) Rate zonal centrifugation:
 In rate zonal centrifugation the solution has a density gradient.
 The sample has a density therefore greater than all the layers the solution.
 The sample is applied in a thin zone at the top of the centrifuge tube on a density
gradient.
 Under centrifugal force, the particles will begin segmenting through the gradient.
 The particles will begin segmenting in separate zones according to their size, shape
and density.
 It is also called velocity centrifugation
B) Isopycnic or sedimentation equilibrium centrifugation:
 In this type of centrifugation, the solution contains a greater range of densities.
 The density gradient contains the whole range of densities of the particle in the
sample pool stop each particle with sediment only to the position in the centrifuge
tube at which the gradient density is equal to the phone density.
 In sedimentation centrifugation separation of particles occur into the zone based on
their density difference, independent of time.
 The solution of the biological sample and cesium salt is uniformly distributed in a
centrifuge tube and rotated in an ultracentrifuge.
 Under the influence of centrifugal force, the cesium salts redistribute to form a
density gradient from top to bottom.
DIFFERENTIAL CENTRIFUGATION
 Differential centrifugation is one type of centrifugation procedure where components
are separated and placed into the centrifuge tube using an increasing centrifugal
forces.
Principle of Differential centrifugation
  Differential centrifugation is based on the variations in the rate of sedimentation of
biological particles that vary in dimensions and densities.
 When the greater force of centrifugal forces is applied, the initial sedimentation of
the bigger molecules begins.
 The particles continue to settle, based on the speed and length of the individual
centrifugation processes as well as the density and relative size of the particles.
Steps of Differential centrifugation
 A homogenized sample is created the buffer that contains it.
 The sample is then put inside the centrifuge tube which is run at the same
centrifugal force for the specified amount of time at a specific temperature.
 After this process the pellet will have formed in the middle of the tube. It is then
separated by the supernatant.
 The supernatant is then added to the new centrifuge tube, where it is then
centrifuged at a new speed for a specific period of period of time and at a specific
temperature.
 In addition, the supernatant gets separate from the pellets that have formed.
 This process continues until the components are isolated from one the other.
 The particles are identified by looking for specific indicators that are specific to the
particular particle.
Uses of Differential centrifugation
 Differential centrifugation is a common method to separate membranes and
organelles of cells within the cell.
 It is also used to separate the nucleus.
 Because this method is able to separate particles based on the size it can be used
to purify extracts that have larger impurities.
PREPARATIVE CENTRIFUGATION
 Concerned with the actual separation, isolation, purification (eg. Whole blood cells,
Subcellular organelles, Plasma membrane, Polysomes, Ribosomes, Chromatin,
Nucleic acid, Lipoproteins and Viruses).
 This is most commonly used technique.
 Separation of one type of particles from other particles. For example: cellular
organelles
 It is commonly used to purify the substances so that further studies can be
performed upon them.
 Divided into two subtypes – differential and density gradient.
 Depends on centrifugal force, shape and size of particles and their density.
REFRIGERATED CENTRIFUGE
 Centrifuges that are refrigerated that come with temperature controls that span
from -20degC up to 30degC.
 Different type of centrifuge is available with the control of temperature that is crucial
for different processes that require lower temperatures.
 Refrigerated centrifuges are equipped with an instrument for controlling
temperature as well as racks and rotors for the test tubes.
 These centrifuges offer an RCF that can reach 60,000 rpm which is perfect for the
separation of different biological molecules.
 They are commonly employed to collect substances which are rapidly separated, like
the yeast cell, chloroplasts and erythrocytes.
ANALYSIS OF SUBCELLULAR FRACTIONS
 Subcellular fractionation Differential centrifugation is used to separate the
components of the cell homogenate.
 Separation is based primarily on differences in the size of the particles.
  Initially, low rotor speeds are used to sediment the largest particles.
 Subsequently, the rotor speed is increased in a step-wise manner, to sediment
particles of other size ranges

APPLICATIONS IN BIOLOGICAL SCIENCES

 To separate two miscible substances
 To analyze the hydrodynamic properties of macromolecules
 Purification of mammalian cells
 Fractionation of subcellular organelles (including membranes/membrane fractions)
Fractionation of membrane vesicles
 Separating chalk powder from water
 Removing fat from milk to produce skimmed milk
 The clarification and stabilization of wine
 Separation of urine components and blood components in forensic and research
laboratories.
 To separate cellular and subcellular components
 Separating one cell type from another.
 Isolating viruses and macromolecules, including DNA, RNA, proteins, and lipids or
establishing physical parameters of these particles from their observed behavior
during centrifugation.
THE BASICS OF THE MICROSCOPE
1. The word microscopе is derived from the Greek word mikros which means small
and skopein which means to look at and the science of exploring small objects
applying such an apparatus is known as microscopy.
2. A microscope is an optical instrument that uses a lens or a combination of lenses
to magnify and resolve the fine details of an object.
3. A microscopе is well-defined as an optical apparatus that uses an arrangement of
lenses to yield an enlarged image of small objects.
4. The earliest methods for examining physical evidence in crime laboratories to study
the structure and composition of matter.
5. The microscope is still one of the most used tools in a crime laboratory for analysing
evidence, even with the advent of other modern instrumentation.
6. The earliest and simplest microscope was the single lens commonly referred to as a
magnifying glass. The handheld magnifying glass makes things appear larger than
they are because of the way light rays are refracted, or bent in passing from the air
into the glass, and back to the air.
7. The magnified image is observed by looking through the lens.
THE SCOPE OF MICROSCOPY IN FORENSIC SCIENCE
 At a suspected crime scene police officer, scene of crime officers (SOCOs), or crime
scene investigators (CSI) may seek evidence of a crime being committed, cause of
death, evidence of involvement, identification of victim, evidence of third-party
involvement, and identification of victims and third parties.
 Advanced microscopy techniques can aid these forensic science investigations
across a wide range of disciplines.
 Evidence of the time since death, the post-mortem interval (PMI), can be of crucial
significance. This can be estimated from macro-observations; however, electron
microscopy can aid examination of the body providing better PMI estimation.
 Characterization, identification, and comparison of any physical evidences.
 Various samples: drug, paint, soil, minerals, dusts, glass, polymers, fibres
(synthetic and natural), paper, wood, hairs, pollens, etc.
 Limited chemical information: polarized light source, fluorescence labelling, coupled
with infrared spectroscopy, and microprobe analysis (electron microscopy)
 PMI can also be estimated from colonization of the body from insects such as
blowflies.
 The stage of development of immature insects can provide evidence of the time of
colonization, and potential time of death.
 The examination and age estimation can be improved through electron microscopy
examination of the morphology of the instar stages.
 The developmental period, adjusted for temperature is species dependent and SEM
can aid identification of closely related species, for example through
ultramicroscopic examination of male genitalia of blowflies.

 Microscopes have been around for the ages.

 Roman philosophers had mentioned “burning glass” within their works.
 However, the first microscope of this type was not invented until the 13 th century.
 Two lenses were set on opposite sides of the tube. This tube of magnifying power
was the basis for our modern-day microscope.
DIFFERENT TYPES OF MICROSCOPY
Type of Microscopes
There are present mainly 2 types of Microscopes. They are classified based on their
working principle and uses.
1. Optical Microscopes: Optical Microscope Also Known as Light Microscope.
A. Simple microscope
B. Compound Microscope
C. Comparison microscope
D. Phase contrast microscope
E. Stereoscopic/Stereo zoom microscope
F. Polarizing microscope
G. Fluorescence microscope
H. IR microscope
2. Electron microscopes: There are two main types of electron microscope;
1. The Transmission Electron Microscope (TEM)
2. The Scanning Electron Microscope (SEM)
SIMPLE LENSE/SIMPLE MICROSCOPE
1. Simple Microscope refers to those microscopes consist of a single lens to enlarge an
object through angular magnification alone, giving the viewer an erect enlarged
virtual image. it cannot be projected onto a screen.
2. These types of microscopes are used different types of lense for magnification such
as; magnifying glass, loupes, and eyepieces.
3. A Simple Microscope is a type of optical Microscope or light Microscope. This was
the first microscope ever created. It was invented by Antony van Leeuwenhoek in
the 17th century. He combined a convex lens and a holder for specimens.

PRINCIPLE OF A SIMPLE MICROSCOPE

 A simple microscope is also called a magnifying glass because of its convex lens of
small focal length. It is used to see the magnified image of an object that is not
visible to the human eyes.
 If you place a tiny object within the focus of the simple microscope, a magnified
image of the object is formed making it easier for the naked eye peeping through the
lens to see the image.
 1. A small object AB which is to be magnified is placed between the principal focus F’
and optical center C of the convex lens.
2. Now, a ray of light AO parallel to a principal axis which is coming from point A of
the object passes through the focus F along the straight line OX after getting
refracted by the convex lens.
3. A second ray of light AC coming from the point A of the object passes through the
optical center C of the convex lens along the straight line CY.
4. As is clear from the figure that the two rays i.e. OX and CY are diverging rays so
these rays can intersect each other only at point A’ when produced backward.
5. Now, on drawing A’B’ perpendicular from point A’ to the principal axis, we get the
image A’B’ of the object which is virtual, erect, and magnified.
MAGNIFICATION OF A SIMPLE MICROSCOPE
The magnifying power of simple microscopes is given by:
It should be noted,
 Smaller the focal length of the lens, greater will be its magnifying power.
 This microscope has a maximum magnification power of 10, which means the
specimen will appear 10 times larger by using the simple microscopes of maximum
magnification.

COMPOUND MICROSCOPE
 A compound microscope is a laboratory instrument with high magnification power,
which is consists of more than one lenses.
 Compound Microscopes are used for the study of structural details of a cell, tissue,
or organ in sections.
 Zacharias Janssen, a Dutch spectacle maker, is credited with inventing the
compound microscope around 1590, according to historians ( more history here ).
 A compound microscope can magnify the image of a tiny object up to 1000.
 The term compound means “multiple” or “complex”.
 The compound microscopes is consists of two lenses includes, the microscope
objective lens (typically 4x, 10x, 40x or 100x) in a rotating nosepiece closer to the
specimen, and the eyepiece lens (typically 10x) in the binocular eyepieces.
 A compound binocular microscope is more commonly used today.
 Zacharias Jansen created a compound microscope that used collapsing tubes and
produced magnifications up to 9X.
 Compound microscopes are generally types of bright field microscope.
 Compound microscopes may be categorized as an upright microscope, and Inverted
microscope.
 Upright compound microscopes are just like an ordinary microscope which has a
lens system, followed by the stage where the specimen is kept, and then the light
source.
 An upright microscope is an inverted microscope that uses two sets (a compound
lens system), which provides higher magnification than a stereo microscopy.
 Inverted compound microscopes are exactly the reverse replica of the upright
microscope with the illumination system first, followed by the stage, and then the
lens system.
  WORKING PRINCIPLE OF COMPOUND MICROSCOPE
 The compound microscopes are works on the principle that when a tiny specimen
to be magnified is placed just beyond the focus of its objective lens, a virtual,
inverted and highly magnified image of the object are formed at the least distance
of distinct vision from the eye held close to the eyepiece.

MAGNIFICATION POWER OF COMPOUND MICROSCOPE

The total magnification level of image formed by the compound microscopes is calculated
b this following formula;
m = D/ fo * L/fe
Where,
 D = Least distance of distinct vision (25cm)
L = Length of the microscope tube
fo = Focal length of the objective lens
fe = Focal length of the eye-piece lens
Use of Compound microscope
1. Compound Microscopes used for blood analysis in pathological labs.
2. In forensic laboratories, the compound microscopes are used for examining the
human cells, paper, etc. Which are related to the crime scene.
3. Used for the detection of drugs, by viewing their particles under compound
microscopes.
4. In university and college laboratories students use compound microscopes for
studying the fungi, bacteria, plant cells, animal cells, etc.
Advantages of Compound Microscopes
• It is not very expensive.
• Can look at live samples
• Can magnify up to 2000 times
• This microscope is easy to use.
• These microscopes are easily transferable due to their compact size.
• It can produce a clear image as compared to a simple microscope.
COMPARISON MICROSCOPE
 In 1925, Major Calvin [Link] wrote an article for the army ordinance title
“Forensic Ballistics”. In it he described the use of the comparison microscope in fire
arms investigations.
 He is generally created with the conception of term “Forensic Ballistics”.
 Philip O. Gravelle developed the comparison microscope for the identification of fired
bullets and cartridge cases with support and guidance of Calvin [Link].
 The comparison microscope is a device used to analyze side by side comparison of
two materials under the same optical conditions.
 The bridge connects the two identical microscopes and allows a split field of view
that permits two separate objects to be viewed simultaneously.
 This avoids the observer having to rely on memory when comparing two objects
under a conventional microscope.
 The primary use of this type of instrument is criminology as in ballistics. Other
scientific fields including paleology and archeology utilize these special comparison
microscopes.

WORKING PRINCIPLE OF COMPARISON MICROSCOPE

 The idea behind the comparison microscope is simple.
 Two microscopes are placed next to each other and the optical paths of each
microscope are connected together by the optical bridge.
  The optical bridge consists of a series of lenses and a mirror that brings the two
images back together at the single eyepiece.
 The user looks through the eyepiece as with a regular microscope, except that a line
in the middle separates the circular view field into two parts.
 The left side of the view field is the image produced by the left microscope, and the
right side of the view field is the image produced by the right microscope.
 In some more modern or sophisticated comparison microscopes, it is also possible
to super-impose the view fields generated by the two microscopes.
 Comparison microscopes are mostly used in a reflected light setting, but a
transmitted light setting is also available in some instances, and fluorescent light
settings are found on higher-end models.
 Reflected light instruments are used by firearms examiners to compare rifling
marks on bullets as well as ejector marks and firing pin impressions on cartridge
cases.
 Tool marks and cut and polished layered paint chips can also be compared with the
same equipment.
 Transmitted light microscopes are used to compare hairs, fibers and layered paint
chips which have been thin-sectioned.
 Polarizing and fluorescence equipment may added to a comparison microscope
which is to be used for fiber comparisons to enhance its capabilities.
 Human hair comparisons, particularly the final stages of an examination, are
conducted almost exclusively under a comparison microscope.
FORENSIC APPLICATIONS OF COMPARISION MICROSCOPE
1. Forensics Examination of Spent Cartridges.
2. Micro-stamping Gun Firing Pins.
3. Forensics Examination of Bullets:
A. According to class characteristics The three main class characteristics of all bullet
are
a) The lands and grooves
b) The caliber of the bullet, and
c) The rifling twist
B. According to individual characteristics ~ by individual striation mark on bullet
Firing pin mark (Individual to each gun)
Left on cartridge when firing pin strikes the bottom of cartridge
Breech mark, Extractor, or ejector mark
Rifling marks
Land and groove marks, Rifling Twist
Striation marks on bullet
Limitations
 The device is very costly.
 It requires very accurate handling.
 The person using the device must be an expert.
THE POLARIZING MICROSCOPE
 Polarizing Microscope( Petrological microscope): In the year (1768-1851), William
Nicol discovered polarizing Microscope.
 It is used in the study mainly for the identification of optical properties of the given
specimen, which is in the form of thin sections of the identifying and comparing
microscopic particles, crystals and fibers, rocks and minerals etc.
Polarized Light?
  Light is an electromagnetic wave. Although light waves can vibrate in all directions,
in general they are described as vibrating in two directions at right angles to each
other.
 Any light which vibrates in more than one direction is called “unpolarized light”
whereas, a light wave that vibrates in a single direction is called polarized light.
 In polarizing Microscope, a polarizer is used which allow passage of light of one
vibration direction.
 The human eye is not sensitive to the direction of vibration of light.
WORKING PRINCIPLE OF POLARIZING MICROSCOPE
 Polaroid is an optical filter which can convert unpolarized light into plane polarized
light ( i.e. light source of bulb, natural light, etc.).
 This polarized light falls on refracting specimen which generates two wave
components that are right angles to each other. These two waves are called ordinary
and extraordinary waves.
 These waves pass through the specimen in different phases. They are recombined
using constructive and destructive interface, by an analyzer. This leads to the final
generation of high-contrast image.

STEREO MICROSCOPE
 During the mid-nineteenth century, Francis Herbert Wenham of London designed
the first truly successful stereomicroscope.
 The stereo microscope, also called a dissecting microscope, as it allows the operator
to manipulate/dissect the specimen while it is being observed through the
microscope.
 It provides relatively lower magnification usually below 100x.
 They provide a close-up 3-Dimensional view of objects surface textures.
 It uses two separate optical paths with two objectives and two eyepieces to provide
slightly different viewing angles to the left and right eyes.
WORKING PRINCIPLE
 The working principle of optical binocular stereomicroscopy depends on two light
paths traveling through the objective lens and ocular lens.
  Two specially separated optical path focuses sample on the same point from slightly
different angles.
 Each light path provides a different angles (usually between 10 and 12 degrees) of
viewing in each of our eye.

USES OF STEREO MICROSCOPES

Due to their ability to view small things in 3D, stereo microscopes can be used in a variety
of applications, especially when the operators need to see and manipulate tiny objects in
real-time.
 Surgery – Precise microsurgery, especially operating in the brain, requires a medical
stereomicroscope.
 Pathology – Pathologists use stereo microscopes to examine skin conditions.
 Biological research – Essential tools to aid in dissection and observation, for
example, Entomology and Botany.
 Paleontology – Paleontologists use stereo microscopes when they clean and analyze
fossils.
 Art and jewelry appraisal – Stereo microscopes can be used to evaluate the value of
artworks.
 Inside the head of a stereo microscope, there are more optical components that we
can’t see. Generally, there are two types of optical systems in stereo microscopes:
the Greenough optical system and the Common Main Objective (CMO) optical
system.
 Greenough-type stereo microscopes have two completely separate optical paths.
There are two objective lenses to pair with the two eyepiece lenses in the cylindrical
cone of stereo microscopes. Two light paths strike the object with a difference in
angle of 15º. The human brain is able to generate a 3-D image through the optical
path.
 CMO or “Parallel style” stereo microscopes do not have double lenses; instead, the
microscope has only one objective lens with a large diameter through which the light
paths run for both the left and the right eye. This is why we called it “Common Main
Objective”. CMO style are more flexible, but also more expensive. This kind of
microscope is better suited to microphotography than the Greenough microscope.
THE PHASE CONTRAST MICROSCOPE
 The first phase contrast microscope was developed by Fritz Zernike in 1933, hence
also referred as Zernike microscope.
 It enables to differentiate transparent, unstained, living (without killing or altering
the living components) structures.
 It is used primarily in serological and glass examinations. Its principal use in
serology is to observe cells in biological fluids.
 Under these conditions cells exhibit a very small optical path difference with respect
to the medium in which they are immersed.
WORKING PRINCIPLE OF PHASE CONTRAST MICROSCOPE
1. The light rays enter the annular diaphragm from its source.
2. Then it passes through the condenser lens, which focused the rays on the specimen.
3. The light transmitted through the specimen and then enter the objective lens where
an image of the specimen is created.
4. As the light transmitted through the specimen it creates a deviated and un-deviated
light rays.
5. The deviated light rays miss the phase ring over the objective lens.
6. Whereas the un-deviated light rays strike a phase ring. As a result, deviated and
un-deviated rays formed different images.
7. The un-deviated light rays formed the background of specimen’s image.
APPLICATIONS OF PHASE CONTRAST MICROSCOPE
1. Phase contrast microscopy is specially useful for the detection of bacterial
components such as endospore and inclusion bodies.
2. Phase contrast microscopy also are widely used in studying Eukaryotic cells.
3. It also used to visualize a thin tissue slice.
Advantages
1. Living cells are observed in the natural state. It will provide more information
about the specimen than specimens that need to be killed, fixed or stain to view
under a microscope.
2. It produces a resolution and high contrast images of the specimen.
3. It is useful for studying on thin specimen.
4. The modern form of this microscope can capture photos or can record videos.
5. Phase Contrast Microscopy can create a visible image of a highly transparent
objects.
6. No need for staining and fixation of the specimen.
 7. Intercellular components of living cells can be observed with high resolution.
DISADVANTAGES/LIMITATIONS OF PHASE CONTRAST MICROSCOPE
1. The Annuli or rings of phase contrast microscope limit the aperture to some
extent, which decreases the resolution of image.
2. Thick organisms or specimens can not be observed by this microscope, a distorted
image can appear.
3. With the uses of white or green lights Images appear as grey or green, which
results a poor photomicrography.
4. Shade-off and halo effects can occur in this microscope.

FLUORESCENCE MICROSCOPY
 A fluorescence microscope is a type of optical microscope that uses fluorescence
reflection and absorption to study properties of organic or inorganic substances.
 Fluorescence refers to the emission of light rays from an exciting substance, which
is excited by UV.
 When some molecules absorb Radiant energy, they become excited and later release
much of their trapped energy as light. This light is called fluorescence light, which
emitted very quickly by the excited molecule. using this technology fluorescence
microscope is Discover.
 In 1852, British scientist Sir George G. Stokes first described fluorescence.
 Eric Betzig, William Moerner, and Stefan Hell developed super-resolved
fluorescence microscopy and they got Nobel Prize On 8 October 2014.
PRINCIPLE OF FLUORESCENCE MICROSCOPE
1. Fluorescence microscope uses a high-intensity Mercury lamp as a Source of
light. This lamp emits white and blue light..
2. The exciter filter transmits only blue lights to the specimen and blocks out all other
colors.
3. The blue light is reflected downward to the specimen by a dichroic mirror, which
reflects the lights of certain colors but transmits light of other colors.
4. The specimen is stained with a fluorescent dye certain portions of the specimen
retains the dye others do not.
 5. The stained portion absorbs blue light and emits green light, which passes upward
penetrates the dichroic mirror and reaches the barrier filter.
6. This filter allows the green light to pass to the eye; however, it blocks out any
residual blue lights from the specimen which may not have been completely
deflected by the dichroic mirror.
7. Thus the eye perceives the stained portion of the specimen as glowing green against
a jet black background whereas the unstained portion of the specimen is invisible.

APPLICATION OF FLUORESCENCE MICROSCOPE

1. The Fluorescence microscope has become an essential tool in medical microbiology
and microbial ecology.
2. Bacterial pathogens can be identified after staining them with fluorescent dye or
specifically labeling them with fluorescent antibodies using immunofluorescence
produce.
3. In ecological studies, the Fluorescence microscope is used to observe
microorganisms stained with Fluorochrome-label probes or Fluorochromes that
bind specific call constitutes.
4. Another Important use of the fluorescence microscope is the localization of specific
proteins within the cell.
ADVANTAGES OF FLUORESCENCE MICROSCOPE
1. It is used to study the dynamic behavior exhibited in live-cell imaging.
2. It can trace the location of a specific protein in the cell.
3. Due to the presence of higher sensitivity, it can detect the 50 molecules per cubic
micrometer.
4. It allows multicolor staining of the specimen.
DISADVANTAGES/LIMITATIONS OF FLUORESCENCE MICROSCOPE
1. During the process of photobleaching, the Fluorophores lose their ability to
fluoresce.
2. Fluorescent molecules can generate reactive chemical species during the
illumination process which enhances the phototoxic effect.
3. The specimen must be stained with the fluorescent dyes.
4. Specimen preparation is a costly process.
ELECTRON MICROSCOPE
  An electron microscope is a microscope which uses electron beams as a primary
source of illumination.
 An EM uses the same principles of an optical microscope but instead of photons or
particles of light, it concentrates electrons, charged particles located on the
outside of atoms, onto the object.
 In 1931, a physicist and electrical engineer, Ernst Ruska and Max Knoll discovered
the first electron microscope.
 It used to obtain high-resolution images of biological and non-biological specimens.
 It is used to study the structure of tissues, cells, organelles, and macromolecular
complexes in detail.
 It has higher resolving power than light microscopes.
 There are two types of electron microscope:
1. Scanning Electron Microscope (SEM)
2. Transmission Electron Microscope (TEM)
SCANNING ELECTRON MICROSCOPE
 A scanning electron microscope uses focused beams of electrons to create an image
of a specimen by scanning the surface area.
 Atoms of specimens are combined with the electron beams and form different types
of signals, which contain data, which are related to the surface topography and
composition of the sample.
 It can produce high-resolution, three-dimensional images of the specimen.
 These types of microscopes are used to examine the surface of microorganisms in
great detail.
 In 1937, Manfred von Ardenne invented the first Scanning Electron Microscope.
PRINCIPLE OF SCANNING ELECTRON MICROSCOPE
1. First of all, an electron source or electron gun located at the top of the SEM column
is heated with high-voltage.
2. As a result, it will release electron beams.
3. Electron beams are now accelerated down the column and onto a series of
electromagnetic lenses.
4. These lenses and tubes are also called solenoids, because they are wrapped in a
coil.
5. These coils create fluctuations in the voltage, which results in
increasing/decreasing the speed of electrons. Thus, how they create focused
electron beams.
6. This electron beam is focused onto a specimen.
7. A computer is attached to the SEM, which controls the magnification power of the
microscope and as well as determines the surface area to be scanned.
8. The electron beams and atoms of the sample are combined, the rate is determined
by the acceleration rate of incident electrons, which carry significant amounts of
kinetic energy before focusing on the sample.
9. When the incident electrons come in contact with the sample, energetic electrons
are released from the surface of the sample. The scatter patterns made by the
interaction yields information on the size, shape, texture, and composition of the
sample.
APPLICATION OF SCANNING ELECTRON MICROSCOPE
1. SEM is used in life science, biology, gemology, and medicine for research purposes.
2. It is also used in forensic laboratories.
3. In industries and technology, it is used for semiconductor inspection, production
line of minuscule products, and assembly of microchips for computers.
4. It is used to examine surface contaminants.
5. It reveals spatial variations in chemical compositions.
6. It provides qualitative chemical analyses and identifies crystalline structures.
7. It provides information in microstructures.
8. It can detect and analyze surface fractures.

ADVANTAGES OF SCANNING ELECTRON MICROSCOPE

1. It provides a 3D and topographical image of the specimen with great detail.
2. Require less time.
3. Require minimal preparation action of the sample.
4. Modern SEMs produces portable digital data.
5. SEM is easy to operate with proper training.
DISADVANTAGES OF SCANNING ELECTRON MICROSCOPE
1. SEMs are a costly item.
2. It is large in size, that is why required a room to operate SEM.
3. The room should be free of electric, magnetic field, and vibration.
4. Required a steady voltage.
5. Required cool water.
6. Required proper training to operate SEM.
7. It is limited to solid, inorganic samples small enough to fit inside the vacuum
chamber.
8. It also carried a risk of radiation exposure.
TRANSMISSION ELECTRON MICROSCOPE
 In a Transmission electron microscope, the electron beam is transmitted through
a very thin specimen or object and forms a highly magnified and detailed image of
the sample.
 The specimen used in Transmission Electron Microscope, should be very thin, less
than 100 nm thick.
 A Transmission Electron Microscope can create a much higher resolution and
magnified image than a light microscope, because of the shorter wavelength of the
electron as compared to photons.
  In TEM the sample’s image is formed by the interaction between the transmitted
electron beam and sample.
 TEM can tell us the structure, crystallization, morphology, and stress of the
specimen in a better way as compared to a simple microscope.
 The formed image is then magnified and visualized on a fluorescent screen (layer of
photographic film).
 Ernst Ruska and Max Knolls discovered the first Transmission Electron
Microscope in 1931.
WORKING PRINCIPLE OF TRANSMISSION ELECTRON MICROSCOPE
 First of all, a tungsten filament is heated, which is also called an electron gun.
 The heated tungsten filament or electron gun will start to release electron beams.
 An electromagnetic coil and high voltage(up to several million volts) applied to these
electron beams to accelerate their speed (extremely high speeds ).
 A condenser lens with a high aperture eliminates all the high angle electrons and
focused all the electron beams into a thin, small beam.
 The high-speed electron beams are now transmitted through the specimen.
 The transmitted electron beams are focused into an image with the help of an
objective lens.
 The electron beams are projected on to a phosphorescent/fluorescent screen, which
creates an image of the specimen, also called a micrograph.
 All the images are captured by a charge-coupled device (CCD) camera, which is
located underneath the screen.

APPLICATION OF TRANSMISSION ELECTRON MICROSCOPE

1. TEM is used to study the topographical, morphological, compositional, and
crystalline information.
2. It helps to analyze the structure and texture of the specimen.
3. TEM used in semiconductor analysis.
4. TEM is also used in production and the manufacturing of computers and silicon
chips.
5. It is also used in Technology companies to identify flaws, fractures, and damages to
micro-sized objects.
6. To distinguish between animal and plant cells.
7. It is also used in nanotechnology for studying nanoparticles like ZnO nanoparticles
 8. It can be used to identify and detect fractures.
9. To study and visualize the cell structures of bacteria, viruses and fungi.
10. View bacteria flagella and plasmids
11. View the sizes and shapes of microbial cell organelles
ADVANTAGES OF TRANSMISSION ELECTRON MICROSCOPE (TEM)
1. TEM provides 1 million times of magnification power as compared to a simple
microscope.
2. It provides detailed information about the structure of specimens.
3. TEM can produce a high-quality and detailed image of the specimen.
4. It is easy to operate with proper training.
5. Permanent images can be produced by it.
6. It can be used in a wide variety of ways, including basic Biology and Nanotechnology,
education, and industrial applications.
LIMITATIONS OF TRANSMISSION ELECTRON MICROSCOPE (TEM)
1. They are very expensive.
2. TEM is very large in size, they require a room to operate.
3. Required proper training to prepare the specimen for TEM.
4. It produces monochromatic pictures unless it uses a fluorescent screen at
visualization’s end.
5. They are sensitive to electromagnetic movements and vibrations, so they are only
used in areas where they are protected.
6. To function, it requires constant voltage flow.
7. They can be difficult to maintain.
8. Artifacts can be dangerously damaged by chemical fixations, dehydrators, or
embedment's.
9. Only electron transparent specimens are used in TEM.
10. To operate a TEM requires special training.
11. It is tedious to prepare specimens for viewing under the TEM.