Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.

1. ELECTROPHORESIS
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2. CONTENTS
 Introduction
 Principle
 Types of electrophoresis
 Factors effecting electrophoresis
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3. Introduction:
 Electrophoresis is the motion of dispersed particles relative to a fluid under the
influence of a spatially uniform electric field
 Positively charged particles – Cataphoresis
Negatively charged particles – Anaphoresis
 It is the basis for analytical techniques used in chemistry for separating molecules by
size, charge, binding affinity
 Separation is achieved by using matrix(gel) or electromotive force to propel the
molecules through the gel
 This technique is used for DNA, RNA, and protein separation
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4. Principle:
 The fundamental principle of electrophoresis is the existence of charge
separation between any surface and fluid in contact with it
 The process involves movement of electrically charged particles in a fluid under
the influence of an electric field where charged particles migrate in the direction
of electrode bearing opposite charge
 The separation effect on the ionic particles results from differences in their
velocity (v), which is the product of the particle's mobility (m) and the field
strength (E):
v=mE
 The mobility (m) of an ionic particle is determined by particle size, shape, and
charge, and the temperature during the separation, and is constant under defined
electrophoretic conditions.
 Electrophoretic conditions are characterized by the electrical parameters
(current, voltage, power), and factors such as ionic strength, pH value, viscosity,
pore size, etc., which describe the medium in which the particles are moving.
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5. Types of electrophoresis
 ZONE ELECTROPHORESIS
 Paper electrophoresis
 Gel electrophoresis
 Thin layer electrophoresis
 Cellulose acetate electrophoresis
 MOVING BOUNDARY ELECTROPHORESIS
 Capillary electrophoresis
 Isotachophoresis
 Isoelectric focusing
 Immuno electrophoresis
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6. Paper Electrophoresis
 In Paper electrophoresis, filter paper is used as a supporting media on which the particles get
migrated
 Filter paper should contain 95% α-cellulose and should have very slight adsorption capacity
 Samples are applied in centre of about 0.5cm diameter, edges of the paper are hanged in
buffer solution.
 After application of sample, electricity is passed to migrate the ionic particles based on the
charge towards the oppositely charged electrodes
 The separation is detected by using various techniques like Ethidium bromide spray, UV
absorption, staining, etc.
 Applications – Serum analysis
Protein analysis
Snake & insect venom analysis
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8. Buffers used in paper electrophoresis
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9. Gel Electrophoresis
 Gel electrophoresis is a method for separation and analysis of macromolecules (DNA,
RNA and proteins) and their fragments, based on their size and charge
 Gels are used as sieving medium
 Gels suppress the thermal convection caused by applied electric field and also serves to
maintain the finished separation so that post electrophoresis process can be applied
 Three types of gel are used. They are Agarose, Starch & Polyacrylamide
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10. Agarose gel electrophoresis
 Agarose gels are made from the natural polysaccharide polymers extracted from
seaweed.
 Agarose gels do not have a uniform pore size, but are optimal for electrophoresis of
proteins that are larger than 200 kDa.
 Agarose gel electrophoresis can also be used for the separation of DNA fragments
ranging from 50 base pair to several megabases (millions of bases).
 Most agarose gels are made with between 0.7% (good separation or resolution of large
5–10kb DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose
dissolved in electrophoresis buffer. Low percentage gels are very weak and may break
when you try to lift them. High percentage gels are often brittle and do not set evenly.
1% gels are common for many applications.
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11. Polyacrylamide gel electrophoresis(PAGE)
 Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins
ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by
the polyacrylamide gel. Pore size is controlled by modulating the
concentrations of acrylamide and bis-acrylamide powder used in creating a gel.
 Care must be used when creating this type of gel, as acrylamide is a potent
neurotoxin in its liquid and powdered forms.
 Typically resolving gels are made in 6%, 8%, 10%, 12% or 15%. Stacking gel
(5%) is poured on top of the resolving gel and a gel comb (which forms the
wells and defines the lanes where proteins, sample buffer and ladders will be
placed) is inserted.
 The percentage chosen depends on the size of the protein that one wishes to
identify or probe in the sample. The smaller the known weight, the higher the
percentage that should be used.
 It is used to separate DNA fragments by single base pairing, protein analysis,
to separate different types of proteins
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12. Starch gel electrophoresis
 Partially hydrolysed potato starch makes for another non-toxic medium for protein
electrophoresis.
 The gels are slightly more opaque than acrylamide or agarose.
 Non-denatured proteins can be separated according to charge and size.
 They are visualised using Napthal Black or Amido Black staining.
 Typical starch gel concentrations are 5% to 10%.
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13.  Page electrophoresis  Agarose gel electrophoresis
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14. Buffer used in gel electrophoresis
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15. Thin layer electrophoresis
 Thin layer electrophoresis is the electrophoretic separation of mixture of amino acids
and peptides on thin layer of silica gel
 Buffer is sprayed on TLC plate evenly and sample mixture is applied as spot or band
on moist layer. Then electric field is applied to separate the mixture of amino acids
based on their electric charge
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16. Cellulose acetate electrophoresis
 Migration takes place on the buffer film on the surface of cellulose acetate plate or
membrane
 Each glucose molecule contains 2-3 acetyl groups, so its adsorption capacity is very
less compared to paper
 It is used in clinical and biological analysis of protein sample(albumins & globulins)
 For staining of sheet after electrophoresis, glycoproteins are used.
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17. Capillary electrophoresis
 Capillary electrophoresis(CE) is separation method performed in submillimeter
diameter capillaries and in micro- and nanofluidic channels.
 In CE method, analytes migrate through electrolyte solutions under the influence of an
electric field. Analytes can be separated according to ionic mobility and/or partitioning
into an alternate phase via non-covalent interactions.
Applications:
 used for the simultaneous determination of the ions NH4+, Na+, K+, Mg2+ and Ca2+
in saliva
 In forensic dept., detection of DNA fragments using Polymerase Chain Reaction(PCR)
 By forensic biologists is typing of STR from biological samples to generate a profile
from highly polymorphic genetic markers which differ between individuals
 Other emerging uses for CE include the detection of specific mRNA fragments to help
identify the biological fluid or tissue origin of a forensic sample
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18. Capillary electrophoresis
• Bubble line detector
• Z line detector
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19. Isotachophoresis
 Isotachophoresis is the technique of selective separation & concentration of ionic
analytes
 It depends on the development of potential gradient
 Leading electrolyte(LE) with high mobility than analyte and Trailing electrolyte with
low mobility is used
 Separation takes place in aqueous medium which contains sucrose to provide density to
the solution
 After application of electric field, a low electric field is created at leading electrolyte
and a high electric field is created at trailing electrolyte
 Separation of the ionic components of the sample is achieved through stacking them
into discrete zones in order of their mobilities, producing very high resolution
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20. Isotachophoresis
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21. Isoelectric focussing
 Isoelectric focussing depends on the development of pH gradient
 PAGE media is used and sample can be placed anywhere in the gel. Current of 2500 V
is applied at 8o C
 High resolution can be achieved permitting separation of proteins differing only by 0.01
Isoelectric point
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22. Immunoelectrophoresis
 The separation and characterization of proteins based on electrophoresis and reaction
with antibodies is called immunoelectrophoresis
 immunoelectrophoresis require immunoglobulins, also known as antibodies, reacting
with the proteins to be separated or characterized.
 Agarose as 1% gel slabs of about 1 mm thickness buffered at high pH (around 8.6) is
preferred for the electrophoresis as well as the reaction with antibodies. The agarose
was chosen as the gel matrix because it has large pores allowing free passage and
separation of proteins, but provides an anchor for the immunoprecipitates of protein and
specific antibodies. The high pH was chosen because antibodies are practically
immobile at high pH..
 Immunoprecipitates may be seen in the wet agarose gel, but are stained with protein
stains like Coomassie Brilliant Blue in the dried gel.
 In contrast to SDS-gel electrophoresis, the electrophoresis in agarose allows native
conditions, preserving the native structure and activities of the proteins under
investigation, therefore immunoelectrophoresis allows characterization of enzyme
activities and ligand binding etc. in addition to electrophoretic separation.
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24. Factors affecting electrophoresis
 Electric field – Voltage
Current
Resistance
 Sample
 Buffer – Composition
Concentration
pH
Heat generation in electric field
 Supporting medium
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25. 1. Electric field:
 The electric field exerts a force on other charged objects and is radially outward from a
positive charge and radially in toward a negative point charge.
 A movement of ions depends upon voltage, current, and resistance of the electric field.
a. Voltage:
 The higher the voltage, the faster DNA will travel through the gel. However,
voltages that are too high can possibly melt the gel or cause smearing or distortion of
DNA bands.
 If the separation of the electrodes is d (meters) and the potential difference between
them is V (volts), the -potential gradient is V/d volts m-1.
 The equation is Vq/d newtons, if force on the ion with a charge is q (coulombs).
 The rate of migration is proportional to Vq/d, so it increases with increase in potential
difference.
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26. b. Current:
 Current is generated due to potential difference applied between the electrodes. It is a
continuous and uniform flow of electrons around a circuit that are being pushed by the
voltage source. It is measured in coulombs sec-1.
 The current is mainly conducted between the electrodes by buffer ions. Thus, increase in
voltage will increase total number of charge towards the electrode. The distance
travelled by the ions is directly proportional to the current and the time.
c. Resistance:
 Electrical resistance is a property of measuring the resistance to the flow of an electrical
current.
 An object of uniform cross-section has resistance proportional to its length, and resistivity
of a material and is inversely proportional to its cross-sectional area.
 Resistance of an electrophoresis unit depends on its size, gel thickness, amount of buffer,
buffer conductivity, and temperature. This resistance normally decreases in time with
increasing temperature. The amount of resistance determines whether the circuit is a
good conductor (low resistance), or a bad conductor (high resistance).
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27.  The resistance, R (measured in ohms, Ω) of an object can be defined as the ratio of
voltage, V (measured in volts) to the current, I (measured in amperes), in accordance
with Ohm’s law,
R = V/I
 The rate of migration of ions is inversely proportional to resistance. Resistance
increases with the length of supporting medium but decreases with its cross-sectional
area and with increase in the buffer ion concentration.
 The power in the supporting medium, W (measured in watts) during electrophoresis is
as shown below.
W= I2/R
 An increase in temperature leads to decrease in resistance. This is due to increase
mobility of ions and evaporation of the solvents from the supporting medium.
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28. 2. Sample:
 Charge, size and shape of the sample being separated affect its own migration rate.
 A net increase in the charge increases the rate of migration. In accordance with
Henderson-Hasselbalch equation, magnitude of charge is pH-dependent.
 Rate of migration is affected by increase in size of molecule (inversely proportional)
and difference in shape of the sample
3. Buffer:
 Buffer affects migration rate of a compound and stabilizes the pH of the supporting medium.
It has been observed that zwitterionic buffers are able to withstand prolonged electrolysis
much better in comparison to the traditional buffers especially in capillary zone
electrophoresis.
a. Composition:
 Most commonly used buffers for the electrophoresis are formate, EDTA, pyridine, Tris,
barbitone acetate and citrate. The buffer should never bind to the molecules being separated
as it effects the migration of the sample. In its simplest form, a buffered solution contains a
mixture of a weak acid and its conjugate base.
HA⇔ H+A–
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29.  The position of acid/base equilibrium is represented by the acid dissociation constant,
Ka. This number is large if the acid is stronger and the equilibrium tends toward
dissociation. While the value is small for an equilibrium that tends toward proton
capture.
 Buffers used in life science range from 10-4 to 10-10 in their Ka values.
Ka = [ H+][A–]/HA
 Where, Ka is usually expressed as its negative logarithm. So, pKa is
pKa= -log[Ka]
b. Concentration:
 Proportion of current carried by buffer increases and the one carried by sample
decreases with the ionic strength of the buffer. Thus, at a low ionic strength the
proportion of current carried by the buffer decreases and those carried by the
sample increases. It leads to overall reduction of current and results in heat production
causing diffusion and loss of resolution.
c. pH:
 The extent of ionization depends on pH, especially in organic compounds. The
ionization increases with increase in pH of an organic compound and its just
reverse for the organic bases therefore affecting its rate of migration. These affects
apply to the ampholytes.
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30. d. Heat generation in electric fields:
 Heating not only changes viscosity and density of the electrophoretic media, it also damages
equipment by warping, cooling blocks, melting plastics, or cracking glass plates. It may also
cause poor resolution and distortion in resolution.
 The generation of heat is given by:
W = E I
Where, W = power in watts
I = current in amperes
 Current and electric field strength are related by the conductivity of the electrophoretic
medium by Ohm’s law, where,
E =I/C
Where, C = medium conductivity (Ω cm-1)
 If the conductivity of an electrophoretic medium is high, electrophoresis becomes
difficult. This is because high conductive solutions result in lower field strength per current
as well as high heat load on the system. This load increases proportional to the current
squared. Electrophoresis is preferred in resistive media and adding polymer particles (such
as, gels) increase the resistivity of media.
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31. 4. The Supporting Medium:
 Migration rate of compounds depends upon type of supporting medium. Inert medium
is always preferred.
 The medium might cause adsorption, molecular sieving, and electro-osmosis –
processes that affect the electrophoretic rate.
 Adsorption causes tailing of the sample, leading to movement of sample in the form of
comet rather than a band. This reduces rate as well as resolution of the separation.
 Molecular sieving is affected by type of gel used.
 Electro-osmosis depends upon the relative charge produced between water molecules in
buffer and surface of supporting material.
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32. THANK YOU
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