1. MOVING BONDARY ELECTROPHOREIS &
ISOELECTRIC PHOCUSING
1
Seminar/Assignment-I
For the Subject
Modern pharmaceutical analytical
techniques (MPH101T)
Submitted to
Drs. Kiran & Pallavi Patel Global
University (KPGU)
Guided by:
Dr. Shailesh Koradiya
Professor
M Pharm. PhD.
KSP
Presented by:
Dhavalkumar Rathod
M. Pharm Sem-I
Pharmaceutics Branch
En. No. 2103313001
Krishna School of Pharmacy & Research (KSP)
Krishna Edu Campus ,Vadodara Mumbai NH#8, Varnama, Vadodara
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CONTENT
Introduction
Definition
Principle and theory of electrophoresis
Factor affecting electrophoresis
Different types of electrophoresis techniques
Zone electrophoresis
Moving Boundary Electrophoresis
Applications of electrophoresis
Disadvantages of Moving Boundary Electrophoresis
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INTRODUCTION
The transport of particles through a solvent by application of an electric field is called as
electrophoresis.
Most of the polymers (containing macromolecules) are electrically charged and will therefore
move in an electric field.
Electrophoresis is useful in identification and structure determination of such big molecules.
DEFINITION
Electro means Electricity and Phoresis means Separation
Separation of serum proteins by the effect of an electric current.
Electrophoresis is a physical method of analysis which involves separation of the compounds
that are capable of acquiring electric change in conducting electrodes.
Electrophoresis may be defined as the migration of the charged particle through a solution
under the influence of an external electrical field.
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Ions that are suspended between two electrodes tends to travel towards the electrodes that bears
opposite charges.
Macromolecules can be characterized by their rate of movement in an electric field.
This property is used to determine protein molecular weights, to distinguish molecules by virtue
of their net charge or their shape and to separate different molecular species quantitatively.
Rate of movement of macromolecules in an electric field is useful parameter to know any
change in amino acid regarding its charge.
Electrophoresis is similar to chromatography.
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Electric field is used as a dragging force.
Technique is simple, very effective and clean.
Large number of samples can be separated, identified and quantitatively measured.
Theory of electrophoresis
As movement of ions or their mobility depends upon the frictional coefficient, which in turn
depends on the function of some of the physical properties of the molecules such as weight,
molecular shape, size etc.
The law of electrostatics states : F electric = qE
where, F electric is electrical force on an ion,
q is the charge on the ion and E is the electric field strength.
The resulting electrophoretic migration of the ion though the solution is opposed by a frictional
force F friction = Vf
where V is velocity (rate of migration) of the ion and f is its ‘frictional coefficient’.
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The working principle of
electrophoresis
The working principle of electrophoresis is that it
causes the separation of the molecules, ions or
colloidal particles that suspends in the matrix under
the force of an electric field.
The electric field allows the migration of the
positively charged molecule towards the anode and
the migration of negatively charged molecule
towards the cathode.
Therefore, electrophoresis is the electrokinetic
phenomenon where the motion of molecules occurs
under an electric field. Factors affecting
electrophoresis
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Strength of electric field
Charge of the sample used
Size of the biomolecule
Binding strength of biomolecule
Hydrophobicity of biomolecule
Shape of the biomolecule
Ionic strength of buffer
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TYPES OF ELECTROPHORESIS
Electrophoresis has two types,
Based on supporting media
(zone electrophoresis)
Without supporting media
Paper electrophoresis
Gel electrophoresis
Cellulose acetate
electrophoresis
Free electrophoresis
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Zone electrophoresis
In this method, an inert polymeric supporting media is used between the electrodes to separate
and analyze the sample.
The supporting media used in zone electrophoresis are absorbent paper, gel of starch, agar and
polyacrylamide.
The major advantage of presence of supporting media is that it minimizes mixing of the sample
and immobilization of the molecule after electrophoresis.
It makes the analysis and purification of the molecule from the gel much easier than the
moving boundary electrophoresis.
The gel electrophoresis is the best example of zone electrophoresis.
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Moving boundary electrophoresis
First used by Swedish biochemist Arne Tisellus, to separate proteins in 1937.
In this method, the electrophoresis is carried in solution, without a supporting media.
The sample is dissolved the buffer and molecules move to their respective counter charge
electrodes.
Moving boundary electrophoresis is carried out in a U shape tube with platinum electrodes
attached to the end of both arms
At the respective ends, tube has refractometer to measure the change in refractive index of the
buffer during electrophoresis due to presence of molecule.
Sample is loaded in the middle of the U tube and then the apparatus is connected to the external
power supply.
Charged molecule moves to the opposite electrode as they passes through the refractometer, a
change can be measured.
As the desirable molecule passes, sample can be taken out from the apparatus along with the
buffer.
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Arne Tiselius (Swedish biochemist)
won Nobel prize In chemistry in 1948 “for
his research on electrophoresis and
adsorption analysis, especially for his
discoveries concerning the complex nature
of the serum proteins”
Only for
information…
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Application of electrophoresis
It is a tool of macromolecular separation
Many biological complex samples can be separated by using various methods of
electrophoresis
In some cases, identification of molecules is also possible
Gel method is more commonly used for routine laboratory experiments as well as research
oriented separations and identification
Electrophoresis is handy tool for biologist and biochemist like the use f chromatographic
techniques by organic chemist.
Many handy instruments are available for conducting such separation experiments
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Disadvantages of Moving Boundary electrophoresis
The resolution of the technique is very low due to the mixing of the sample as well as over-
lapping of the sample components.
The electrophoresis technique is not good to separate and analyze the complex biological
sample instead it can be used to study the behavior of the molecule in an electric field
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Content
Introduction
Isoelectric point (pl)
Principle of isoelectric focusing (IEF)
Capillary isoelectric focusing
Ampholytes
Working procedure
Set up gel
Sample preparation and loading
2D gel electrophoresis
Advantages
Disadvantages
Application
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Introduction
Isoelectric focusing methods are widely applied for the separation of proteins, peptides and
enzymes according to the iso-electric point.
IEF began in 1964, when olaf vesterberg a Swedish patent on the synthesis of new chemical
called carrier ampholytes. The technique was popularized by H. svensson in Sweden.
The principle: In a pH gradient the sample components migrate towards the anode or the
cathode to the pH values, where their net charges are zero: their isoelectric points (pI).
The proteins are driven to their isoelectric points by the electric field.
Basic principle involved is electrophoresis.
Requres a solid surface normally polyacriamide.
Iso-electric
Iso: “same”
Electric:
“charge”
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Iso-electric point (pl)
The PH at which net charge on protein becomes zero is
called iso-electric point.
Below (pl) – positive charge
Above (pl) – negative charge
Protein move toward the electrode with the opposite
charge.
During motion, proteins will either pick or loose
protons.
Different from conventional electrophoresis migrate to
steady state.
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Principle of Isoelectric focusing
All proteins have an isoelectric point PH.
A procedure to determine the isoelectric
point of proteins thus, a mixture of proteins
can be electrophorized through a solution
having a state PH gradient in form the
anode to the cathode and a each protein
will migrate to the position in the PH
gradient according to its isoelectric point.
This is called ISOELECTRIC
FOCUSING.
Protein migrate into the point where its net
charge is zero-isoelectric PH.
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Capillary isoelectric focusing
PH gradient.
Sample focusing and detection.
Not useful for chiral compounds.
Movement of gradient towards the detector.
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AMPHOLYTES
Establishment of stable PH gradient is important.
Achieved by means of commercially available synthetic carrier amphoteric electrolytes.
600-900 Da.
Closely spaced pl and high conductivity.
The curve is determined by PH interval covered by the ampholyte and the distance between
electrodes.
WORKING PROCEDURE
Following chemicals required-
Acrylamide solution, water, ampholyte solution PH 3.5-10; ampholyte soluton PH 4-6.
Urea, distilled water.
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Spin gently to mix urea
Add 10% APS and TEMED at the end.
Remove bubbles.
Fill the cassette completely with solution.
Allow to polymerize at room temperature.
Centrifuge the sample to remove any aggregate.
Apply the supernatant to the wells with a disposable tip or Hamilton syringe.
Run the gel at 150v for 30 min and then at 200v for 2hr.
SET UP GEL
Remove the comb carefully after gel has polymerized.
Attach gel to the electrophoresis tank according the instructions of manufacturer.
Add catholyte (sodium hydroxide) to the upper buffer chamber and anolyte (phosphoric acid)
to the lower buffer chamber.
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SAMPLE PREPARATION AND LOADING
Mix protein with equal volume of 2x loading buffer
Loading buffer includes the following reagents-
-Urea -Ampholyte solution PH 3.5-10.
-Ampholyte solution PH 4-6. -Triton X-100.
-2-mercaptoethanol. -1% bromophenol blue.
2D GEL ELECTROPHORESIS
Technique of IEF and SDS PAGE combined.
Protein separated in two dimension.
1. On the base of pl.
2. On the base of basis of molecular weight in normal SDS PAGE.
Procedure can be adapted by combining IEF and PAGE.
Series of spots formed in gel.
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ADVANTAGES
Proteins that by as little as 0.001 PH units can be separated.
As spreading of bands is minimized due to application of the applied field and the PH gradient,
high resolution can be achieved
Isoelectric focusing (IEF) is a powerful analytical tool of the separation of proteins.
Performing IEF is easier because the placement of sample application is not important.
DISADVANTAGES
Limited stability of solutions.
Lot-to-lot inconsistency.
Inadequate purity for application as a standard.
Carrier ampholytes are generally used in high concentration, a high voltage (upto 2000v) is
necessary. As a result the electrophoretic matrix must be cooled which sometimes makes it
difficult.
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APPLICATIONS
For separating proteins and peptides.
Used in limit test when the density of band is compared with the density of band of std
preparation.
For research in taxonomy, cytology and immunology etc.
IEF gel is used as identity test when migration pattern on gel is compared with std preparation.
Isoelectric focusing (IEF) offers an effective alternative to conventional electrophoresis for
genetic marker typing.
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