ANALYTICAL TECHNIQUES IN BIOCHEMISTRY AND BIOPHYSICS FOR MACRO MOLECULES

A
SEMINAR ON
BY-
Dr. ARUNIMA KARKUN
ASST. PROFESSOR
G.D. RUNGTA COLLEGE OF SCIENCE AND TECHNOLOGY
KOHKA- KURUD ROAD, BHILAI.
ANALYTICAL TECHNIQUES IN BIOCHEMISTRY AND BIOPHYSICS FOR MACRO
MOLECULES

INTRODUCTION
 VARIOUS ANALYTICAL TECHNIQUES
DIALYSIS
PAPER CHROMATOGRAPHY
ION-EXCHANGE CHROMATOGRAPHY
 GEL FILTRATION CHROMATOGRAPHY
 AFFINITY CHROMATOGRAPHY
 HPLC
 PAPER ELECTROPHORESIS
 GEL ELECTROPHORESIS
 ISOELECTRIC FOCCUSING
UV-VISIBLE SPECTROSCOPY
 MASS SPECTROSCOPY
 CONCLUSION
 SUMMARY
 REFERENCES
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ANALTICAL TECHNIQUES IN BIOCHEMISTRY AND BIOPHYSICS FOR MACRO
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Biochemistry is not pure, but an applied discipline & is both analytical & quantitative
to explain the cause & effect of relationship in molecular terms.
 Analysis means getting to bottom of things.
In biochemical studies , experimental models are first subjected to qualitative analysis
i.e., the complex system is broken down & constituents are separated & identified.
Definition :
An analytical technique is a method that is used to determine the
concentration of a chemical compound.
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ANALTICAL TECHNIQUES IN BIOCHEMISTRY AND BIOPHYSICS FOR MACRO MOLECULES
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 There are variety of analytical techniques availabel for the quantification of
macromolecules such as :
 Dialysis
 Paper chromatography
 Ion exchange chromatography
 Affinity chromatography
 Paper electrophoresis
 Isoelectric focusing
 Gel electrophoresis
 UV-Visible spectroscopy
 Mass spectroscopy 3

INTRODUCTION:
 In this process small solutes diffuse from a
high concentration solution to a low
concentration solution across a semi permeable
membrane until equilibrium is reached.
PRINCIPLE:
 The porous membrane selectively allows
small solute to pass while retaining larger
species.
 It is a separation process based on size
selection.
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PROCEDURE:
 This method is specifically used for separation of polysaccharides & lipoproteins.
 In dialysis a porous membrane is present which allows only the selective
molecules to pass through it & macromolecules are left behind.
 The dialysis membrane consists of a spongy matrix of cross linked polymers, the
pore rating referred to as indirect measure of the retention performance.
 Since a solute’s permeability us also dependent upon molecular shape, degree
of hydration , ionic charge & polarity, it is recommended to select a MWCO that
is half the size of the molecular weight of the species to be retained or twice the
size of the molecular weight of the species intended to pass through
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INTRODUCTION :
This technique is commonly used for the separation of amino
acids, sugars, sugar derivatives & peptides.
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 :
 In paper chromatography a few drops of solution containing a mixture of the
compounds to be separated is applied (spotted) at one end, usually ~ 2 cm above a
strip of filter paper.
 The paper is dried & dipped into a solvent mixture ( for amino acid a mixture of
butanol, acetic acid & water in 4:1:5 ratio commonly called BAW).
 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. 7

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Fig.2:-Technique of paper chromatography
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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
 In some cases , a second run is carried out by a different solvent
system, in a direction perpendicular to the first run. This is referred
to as two- dimensional chromatography.
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Introduction:-
Ion exchange chromatography is used for separation of amino
acids, proteins, nucleotides.
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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Procedure :-
 An anion exchanges (R⁺ A⁻) exchanges its anion (A⁻) with another anion
(B⁻) in solution.
R⁺ A⁻ + B⁺ = R⁺ B⁻ + A⁻
 A cat ion 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.3:- Ion exchange chromatography
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For protein purification two common cellulose derivatives, carboxymethyl cellulose
(CMC,cationic derivative) & diethyl aminoethyl cellulose (DEAE, anionic derivative) are
used.
 An even more versatile substances for the separation of protein is a crosslinked dextrin
called sephadex.
When packed in a column & equilibrated with buffer, the polysaccharide acts as a
molecular serve for protein molecules. In these proteins are given negative charge
treating with SDS.
 Thus, electrostatic attraction of oppositely charged ions on a polyectrolyte surface forms
the basis of exchange chromatography.
 The protein mixture is passed through a column of the modified cellulose contained in a
glass tube & allowed to become to the column material.
 Increasing salt concentration & buffer of varying pH values are then passed through
columns.
Protein will be washed off the column at different times depending on their
molecular structure, net charge & side group.
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Introduction :-
 It is used for the separation of both smaller & bigger molecules.
Its apparatus consists of a column packed with sponge like gel beads (usually cross-
linked polysaccharides ) containing pores.
This technique is also referred to as molecular sieves or molecular exclusion
chromatography.
Principle :-
In this technique the separation of molecules is based on the size, shape & molecular
weight.
Procedure :-
 The gel serves as molecular sieves for the separation of smaller & bigger molecules.
 The solution mixture containing molecules of different size (say protein) is applied to
column & eluted with a buffer.
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The larger molecules cannot pass through the pores of gel & therefore move faster.
 On the other hand, the smaller molecules enter the gel beads & are left behind
which come out slowly.
 By selecting the gel beads of different porosity, the molecules can be separated.
 The commercially available gel include sephadex (G-10, G-25, G-100), bio gel (P-
10, P-30, P-100) & sepharose (6B, 4B, 2B).
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Fig:4:-Gel Filtration Chromatography
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Introduction :-
 Affinity chromatography is useful for purification of enzymes, vitamins, nucleic acids,
drugs, hormones, receptors, antibodies, etc.
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. 17

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Fig:5:-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.
PRINCIPLE:
 It may employ the principles of adsorption, partition,
ion-exchange, exclusion & affinity chromatography.
This makes it an extremely versatile technique.
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FIG.6:-outline of HPLC
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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 :-
It is used for the separation of serum proteins.
Principle :-
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.7;- process of paper electrophoresis
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Introduction :-
 It is used for the separation of proteins & nucleic acid.
Principle :-
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. 24

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 The sample components get separated on the basis of their size & electric
charge.
 The separated components can be identifie either by labeling with a radio
isotope or by UV- visible spectrophotometer.
FIG.8:- GEL GLECTROPHORESIS
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Introduction :-
 It is used for purification of proteins.
Principle :-
 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.
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Fig.9:- Isoelectric focusing
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INTRODUCTION:
 Ultra-violet visible spectroscopy refers to absorption spectroscopy or reflectance
spectroscopy in the ultraviolet-visible spectra.
Principle:
 It uses the visible & adjacent ranges. The absorption or reflectance in the visible range
directly affects the perceived colour of the chemicals involved.
 In this region of electromagnetic spectrum , molecules undergo electronic transitions.
The absorption measures transition from the ground state to the excited state.
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Fig:10:UV-VIS Spectroscopy
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PROCEDURE:
It is used in analytical chemistry for the quantitative determination of different
analytes such as transition metal ion, organic compounds & biological
macromolecules.
Determination is usually carried out in solution. Organic solvents may have
significant UV absorption.
The Beer-Lambert law states that the absorbance of a solution is directly
proportional to the concentration of the absorbing species in the solution & the
path length.
It is necessary to know how quickly the absorbance changes with concentration.
This can be taken from references or more accurately , determined from a
calibration curve.
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INTRODUCTION:
 A mass spectrometer is an instrument that can measure the mass/charge ratio(m/z)
of ions in a vaccum.
 From these data , molecular masses can be determined with a high degree of
accuracy, allowing the molecular composition of a given sample or analyte to be
determined .
Principle:-
 Mass spectrometers have three principle components:
a) A source of ions : Converst the analyte into gas phase ions in a vaccum.
b) A mass analyser : Seperates ions according to their mass/charge ratios.
c) Anion detector : Record the impart of individual ions.
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PROCEDURE:
 The analyte is dissolved & forced
through a narrow needle held at a high
voltage.
 A fine spray of charged droplets
emerges from needle & is directed into
the vaccum chamber of the mass
spectrometer through a small orifice.
 As they enter the mass spectrometry ,
the droplets are dried using a stream of
inert gas, resulting in gas phase ions
that are accelerated through the
analyzer towards the detector.
 Because electrospray ionization(ESI)
produces gas-phase ions from solutions
, it is readily integrated with upstream
protein seperation by liquid
chromatography(LC).
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Fig:11:Mass Spectroscopy
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Analytical technique is very important .
 It can be used both for separation & identification of macromolecules &
micro moleculses.
 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.
33

An analytical technique is a method that is used to determine the concentration of a
chemical compound.
 A variety of analytical techniques are availabel for the quantification of
macromolecules.
 Paper chromatography is commonly used for the separation of amino acids, sugars,
sugar derivatives & peptides.
 Ion exchange chromatography is used for separation of amino acids, proteins,
nucleotides.
 Gel filtration chromatography is used for the separation of both smaller & bigger
molecules.
 Affinity chromatography is useful for purification of enzymes, vitamins, nucleic
acids,
drugs, hormones, receptors, antibodies, etc.
 Paper electrophoresis is used for the separation of serum proteins.
 Gel electrophoresis is used for the separation of proteins & nucleic acid.
 Isoelectric focusing is used for purification of proteins.
It is used in analytical chemistry for the quantitative determination of
different analytes such as transition metal ion, organic compounds &
biological macromolecules.
 A mass spectrometer is an instrument that can measure the mass/charge
ratio(m/z) of ions in a vaccum.
 Analytical technique is a very important.
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UPADHYAY,UPADHYAY & NATH 2009 BIOPHYSICAL CHEMISTRY
PRINCIPLES & TECHNIQUES
J.L JAIN 2007 FUNDAMENTALS OF
BIOCHEMISTRY
U.SATYANARAYAN & U.CHAKRAPANI 2008 BIOCHEMISTRY
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