Analytical techniques ppt

A
SEMINAR
ON
ANALYTICAL TECHNIQUES IN BIOCHEMISTRY
PROF. J.P. SHARMA (DIRECTOR)
DR. R.K. RAO (PRINCIPAL)
GUIDED BY - HUMA NAZZ
SIDDIQUI
PRESENTED BY..
NIKITA DEWANGAN
M.Sc.1st SEM
BIOTECHNOLOGY
G.D. RUNGTA COLLEGE OF SCIENCE & TECHNOLOGY
KOHKA-KURUD,BHILAI DURG (C.G.)

 INTRODUCTION
 VARIOUS ANALYTICAL TECHNIQUES
 PAPER CHROMATOGRAPHY
 ION-EXCHANGE CHROMATOGRAPHY
 GEL FILTRATION CHROMATOGRAPHY
 AFFINITY CHROMATOGRAPHY
 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
 PAPER ELECTROPHORESIS
 GEL ELECTROPHORESIS
 ISOELECTRIC FOCCUSING
 SPECTROPHOTOMETER
 CENTRIFUGE
 ENZYME LINKED IMMUNO SORBANT ASSAY
 CONCLUSION
 SUMMARY
 REFERENCES
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Introduction
In analytical biochemistry uses instruments and used to
separate, identify and qualify biomolecules.
Definition
An analytical techniques is a method that is used to
determine the concentration of a chemical compound.
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 Chromatography
 Electrophoresis
 Spectrophotometer
 Centrifugation
 Enzyme Linked Immuno Sorbant Assay
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INTRODUCTION
Chromatography is an analytical technique dealing with the separation of
closely related compound from a mixture. These include protein, peptides,
amino acid, lipids, carbohydrates, vitamins and drugs.
PRINCIPLE
 Chromatography (Greek ; chroma – colour, graphein – to write) usually
consists of a mobile phase and a stationary phase.
 The mobile phase refers to the mixture of substances( to be separate),
dissolved in a liquid or a gas. The stationary phase is a porous solid matrix
through which the sample contained in the mobile phase percolates.
 The interaction between the mobile and stationary phases results in the
separation of the compounds from the mixture.
 TYPES OF CHROMATOGRAPHY - PAPER CHROMATOGRAPHY, ION
EXCHANGE CHROMATOGRAPHY, AFFINITY CHROMATOGRAPHY.
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INTRODUCTION
Paper chromatography is useful for separating the mixture of amino acid,
sugar, chemicals, lipid , urea and some drugs.
PRINCIPLE
 The filter paper are used to support a stationary water phase while mobile
organic phase moves down the suspended paper strip in a cylinder.
 Separation is based on a liquid-liquid partition of the compounds.
 Thus, this is essentially a form of partition chromatography between 2 liquid
phases, although adsorption to the paper may also take place.
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PROCEDURE –
 The sample mixture is applied to a piece of
filter paper the edge of the paper is
immersed in a solvent.
 The aqueous component of the solvent
system binds to the paper & forms a
stationary phase.
 The organic component that migrates on the
paper is the mobile phase.
 When the migration of the solvent is
upwards, it is referred to as ascending
chromatography.
 In descending chromatography, the solvent
moves downwards.
 As the solvent flows, it takes along with it
the unknown substances.
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Fig. 1; paper chromatography

 The rate of migration of the molecules depends on the relative solubility in the
stationary phase (aqueous) & mobile phase (organic).
 The paper (chromatography) is then removed, dried & developed
for the identification of the specific spots.
 Identification of the compound :-
The distance travelled by a particular component of a mixture (or solute)is used
to identify it.
Resolution Factor(RF) = Distance from origin run by the solute
Distance from origin run by the solvent 7
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INTRODUCTION
Ion exchange chromatography is used for separation of amino acids, proteins,
nucleotides and charged molecule.
PRINCIPLE
Ion exchange chromatography involves the separation of molecules on the basis of
their electric charges. Ion exchange resins- cat ion exchanges & anion exchanges are
used for this purpose.
PROCEDURE- An anion exchanges (R⁺ A⁻) exchanges its anion (A⁻) with
another anion (B⁻) in solution.
R⁺ A⁻ + B⁻ = R⁺ B⁻ + A⁻
 A cation exchanges ( H⁺R⁻) exchanges its cat ion (H⁺) with another cat ion ( C⁺) in
solution.
H⁺ R⁻ + C⁺ = C⁺ R⁻ + H⁺
 Thus, in ion exchange chromatography, ions in solutions are reversibly replaced by
ion-exchange resins.
 The binding abilities of ions bearing positive or negative charges are highly pH
dependent, since the net charge varies with pH .
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FIG.:- 2 Ion exchange chromatography
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INTRODUCTION
Affinity chromatography is a method of separating biochemical mixtures based on a
highly specific interaction such as that between antigen and antibody, enzyme and
substrate, Or receptor and ligand.
PRINCIPLE
 The principle of affinity chromatography is based on the property of specific &
noncovalent binding of proteins to other molecules, referred to as ligands. For instance
enzymes bind specifically to ligands such as substrates or cofactors.
PROCEDURE
 This technique involves the use of ligands covalently attached to an inert & porus
in a column.
 The immobilized ligands acts as molecular fish hooks to selectively pick up the
desired protein while the remaining proteins pass through the column.
 The desired protein captured by the ligands, can be eluted by using free ligand
molecules.
 Alternatively, some reagents that can break protein- ligand interaction can also be
employed for the separation.
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Fig:-3 Affinity chromatography
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INTRODUCTION
 High Performance Liquid Chromatography (HPLC) is used to separate
components of a mixture by using a variety of chemical interactions between the
substances being analyzed & the chromatography column.
12Fig; 4. HPLC

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PRINCIPLE
 It may employ the principles of adsorption, partition, ion-exchange, exclusion
& affinity chromatography. This makes it an extremely versatile technique.
PROCEDURE
 The sample to be analyzed is introduced in a small volume to the stream of
mobile phase.
 It is retorted to specific chemical or physical interactions with the stationary
phase. It travels the length of the column.
 The amount of retardation depends on the nature of the analyte, stationary phase
& mobile phase composition.
 At the time at which a specific analyte elutes is called the retention time.
 The use of pressure increases the linear velocity giving the components less
time to diffuse with in the column.
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INTRODUCTION
The movement of charged particles(ions) in an electric field resulting in
their migration towards the oppositely charged electrode is known as
electrophoresis.
TYPES OF ELECTROPHORESIS – PAPER ELECTROPHORESIS,
GEL ELECTROPHORESIS, ISOELECTRIC FOCUSSING.
PAPER ELECTROPHORESIS - Separation of charged particles is determined by
differences in their migration rate which varies with electrical charge, size of
particles & shape of particles.
PROCEDURE -
 In this the sample is applied on a strip of filter paper wetted with dried buffer
solution.
 The end of the strip are dipped into the buffer reservoirs in which electrodes are
placed. Then electric current is supplied allowing the molecules to migrate for
sufficient time.
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 The paper is removed, dried & stained with a dye that specifically colors the
substance to be detected which can be identified by comparing with a set of
standards run simultaneously.
For the separation of serum proteins whatman No.1 filter paper, veronal or tris
buffer at pH 8.6 & the stains amido black or bromophenol blue are employed.
Fig 5, PROCESS OF PAPER ELECTROPHORESIS
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INTRODUCTION
 It is used for the separation of proteins & nucleic acid.
 This technique involves the separation of molecules based on their size, in
addition to electric charges.
PROCEDURE
 In this technique gel is used as a stabilizing media.
 Gel contains wells made with the help of comb.
 Buffer is added in the apparatus.
 In the well the sample is loaded.
 The electric current is switched on.
 The separated components can be identify either by labeling with a radio
isotope or by UV- visible spectrophotometer.
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INTRODUCTION
 It is used for purification of proteins.
 This technique is primarily based on the
immobilization of the molecules at isoelectric
pH during electrophoresis.
PROCEDURE
 Stable pH gradients are set up (usually in gel)
converting the pH range to include the
isoelectric points of the components in a
mixture.
 As the electrophoresis occurs, the molecules
migrate to positions corresponding to their
isoelectric points, gets immobilized & form
sharp stationary bonds.
 The gel blocks can be stained & identified.
Fig -7 Isoelectric focusing

SPECTROPHOTOMETER – Spectrophotometer is a method to measure how much a
chemical absorbs light, by measuring the intensity of light as a beam of light passes
through sample solution.
PRINCIPLE OF SPECTROPHOTOMETER - The principle of spectrophotometer is
based on Lambert’s Beer’s law. :-
1. Lambert’s law (Bouguer’s law) - this law states that the amount of light absorbed is
directly propotional to the length or thickness of the solution under analysis.Thus,
I / I0 = e -kb
 I = the intensity of the transmitted light, I0 = the intensity of the incident light, b = the
absorbing thickness, better known by the term path-length, k = the linear absorption
coefficient of the absorbing material,
 the power term in the above relationship can be removed by converting to the logarithmic
form, thus,
In I /I0 = - kb or In I0 /I = kb,
 Changing the common logarithms we get2.
2.303 Log10 I0/I = kb
2. Beer’s law - this law states that the amount of light absorbed is directly proportional
to the concentration of the solute in solution. Thus,
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2.303log10 I0/I = kʹC
 Where kʹ = absorptivity constant , C = the concentration of the
absorbing material.
 The beer- lambert’s law then becomes:
Log10 I0/ I = abC
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20Fig - 8 spectrophotometer

 CENTRIFUGE is device for separating particles from a solution according
to there size, shape, density, viscosity of the medium.
 PRINCIPLE : - An object moving in a circle at a steady angular velocity will
experience a force F directed outwards. This is the basis of centrifugation. Angular
velocity ω and the radius of rotation ‘r’ in cm collectively determine the magnitude
of the force F,
 F = ω2r
 If F is expressed in the form of gravitational force it is divided by 980.
 RCF = ω2r / 980
 RCF = relative centrifugal force
 But ω = 2π radian = 2π/60 r/min
 RCF = 4π2( r/m)2r/ 3600× 980
 RCF = 1.118 × 15-5 rpm2r
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ENZYME LINKED
IMMUNOSORBANT
ASSAY
 ELISA is based on affinity.
 An ELISA test can
detect both current and
past infections. As well as
antibodies, an ELISA test
can also be used to detect
antigens (substances that
provoke an immune
response, such as bacteria
or proteins) or enzymes.
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Fig ; 9ELISA

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 Analytical techniques in biochemistry provides an opportunity for
researchers and scientists to explore the advanced and latest research
developments in the field of biochemistry .
 Biochemistry deals with the structures, functions and interactions of
biological macromolecules such as proteins, nucleic acids, carbohydrates and
lipids which provide the structure of cells and perform many functions
associated with life.
 It can be used both for separation & identification of macromolecules
& micro molecules.
 In biochemistry, the term macromolecule is applied to the four
conventional biopolymers ( nucleic acids, proteins , carbohydrates & lipids )
as well as non polymeric molecules with large molecular mass.
 The constituent molecules from which macromolecules are assembled are
called monomers, dimers or tetramers.
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 An analytical technique is a method that is used to determine the concentration of
a chemical compound.
 CHROMATOGRAPHY is used to separation of closely related compound from a
mixture.
 Chromatography types – PAPER CHROMATOGRAPHY, ION- EXCHANGE
CHROMATOGRAPHY, AFFINITY CHROMATOGRAPHY, HPLC.
 ELECTROPHORESIS – The movement of charged particle an electric field
resulting in their migration towards the oppositely charged electrode .
 Electrophoresis types – PAPER ELECTROPHORESIS, GEL
ELECTROPHORESIS, ISOELECTRIC FOCUSING.
 Spectrophotometer is the quantitative measurement of the reflection or
transmission properties of a material as a function of wavelength.
 CENTRIFUGE - it is a device for separating particles from a solution according to
their size, shape, density and velocity of medium.
 ELISA - An enzyme-linked immunoassay (ELISA) is a test that detects
antibodies in the blood.
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U. SATYANARAYANA 2010 BIOTECHNOLOGY
J.L JAIN SIXTH EDITION 2010 FUNDAMENTAL
OF
BIOCHEMISTRY
UPADHAYAY FOURTH EDITION2007 BIOPHYSICAL
AND
BIOCHEMISTRY
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