ELECTROPHORESIS

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ELECTROPHORESIS
Doç. Dr. Mustafa Çulha Genetics and
Bioengineering Department
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ELECTROPHORESIS

• Electrophoresis is a separation method based on
  differential rates of migration of charged
  species in an applied dc electric field. Species
  with diffrent charge to mass (e-/m) ratio migrate
  at different velocity under the influence of an
  electric field.
• Swedish chemist Arne Tiselius in 1930s developed
  to study serum proteins and he was awarded the
  1948 Nobel Price for his work.
• Electrophoresis on macro scale has been applied
  to a variety of difficult analytical separation
  problems such as inorganic anions and cations,
  amino acids, catecholamines, drugs, vitamins,
  carbohyrates, peptides, proteins, nucleic acids,
  nucleotides, polynucleotides, and numerous other
  species.

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MODES OF ELECTROPHORESIS

• Capillary Zone Electrophoresis
• Micellar Electrokinetic Capillary Chromatography
  (MECC)
• Cyclodextrin Distribution Capillary
  Electrochromatography (CDCE)
• Capillary Gel Electrophoresis (CGE)
• Native Polyacrylimide Gel Electrophoresis (PAGE)
• SDS-PAGE
• Capillary Isoelectric Focusing (CIEF)
• Capillary Isotachophoresis (CITP)

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CAPILLARY ZONE ELECTROPHORESIS (CZE)
Q number of ionic charges rionic radius,
nsolution viscosity
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A more realistic picture....
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InstrumentationCapillary

• Normally in the 25-100 cm range
• Bore size and thickness of the silica are
  important parameters
• Using a smaller internal diameter and thicker
  walls help prevent Joule Heating, heating due to
  voltage

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Instrumentation Detector

• UV/Visible absorption
• Fluorescence
• Mass Spectrometry

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An electrophoretic Separation
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Electrophoresis

• The movement of ions solely due to the electric
  field, potential difference
• Cations migrate toward cathode
• Anions migrate toward anode
• Neutral molecules do not favor either

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Electrophoresis
The velocity of an ion can be given by
v ion velocity ?e electrophoretic mobility E
applied electric field
The electric field is simply a function of
applied voltage and capillary length (in
volts/cm)
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Electrophoresis
The electric force can be given by
and the frictional force (for a spherical ion) by

• q ion charge
• solution viscosity
• r ion radius
• v ion velocity

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Electrophoresis
During the electrophoresis a steady state due to
the balanced frictional and electric forces
then,
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Electroosmotic Flow (EOF)
A high potential (15 25 kV) is applied across a
capillary column. At pH gt 3, the inner capillary
wall is negatively charged due to ionization of
SiOH. Cations in the buffer adsorb on the walls
leading to an electrical double layer. The
cations are attracted towards the cathode
(negative electrode). Since they are solvated
they drag the solvent molecules with them
resulting in electroosmotic flow of the
mobilephase.
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ELECTRICAL DOUBLE LAYER
Compact inner layer (20 Å) potential
dereases linearly with distance.
Diffuse layer(20-300 Å) Potential
decreases exponentially.
Potential difference across the fixed and diffuse
layersZeta potential (?)
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Electroosmotic Mobility

• where
• ? dielectric constant
• zeta potential
• ? viscosity

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FLOW PROFILES
Electroosmotic Flow Hydrodynamic flow
Electroosmotic flow has a flat profile while
hydrodynamic flow has a parabolic profile. As a
result, CE results in minimal band broadening.
CE efficiencies can be 100,000-200,000 plates/m
compared to 5000-10,000 plates/m in HPLC.
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ELECTROOSMOTIC AND ELECTROPHORETIC MOBILITIES
Electroosmotic flow velocity ? is related to the
electroosmotic mobility ?eo and the field
strength E (V cm-1 Applied voltage/length of
capillary))
The velocity of an ion in the presence of
electroosmosis is the sum of its migration
velocity and the electroosmotic flow.
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Separated bands in CE with cathodic EOF
Neutrals move with electroosmotic flow.
Electroosmotic flow can be reversed by coating
the walls with a cationic surfactant. It can be
eliminated by silanization with trimethylchloro
silane.
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SAMPLE INJECTION
Electrokinetic Injection One end of the
capillary and its electrode are removed from
their buffer compartment and placed in a small
cup containing the sample. A potential is
applied for a certain time causing the sample to
enter the capillary by a combination of ionic
migration and electroosmotic flow. The capillary
end and the electrode are then placed back into
the regular buffer solution. This method injects
more of the faster moving ions than the slower
moving ions.
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SAMPLE INJECTION
Pressure Injection This is achieved by placing
one end of the capillary in a container and
driving the sample into the capillary by
pressurizing or applying a vacuum At the detector
end of the capillary or by elevating the sample
end. Pressure injection does not discriminate
between ions of different mobilities. The volume
of sample injected in CE is 5 50 nanoliters.
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ANALYTICAL PARAMETERS

• Mobility, cm2 / v s
• (1)
• Apparent mobility, µapp
• (2)
• l capillary length to detector, cm
• Vapplied voltage, v
• L capillary total length, cm
• t migration time for species, s

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ANALYTICAL PARAMETERS
Electroosmotic mobility, µEOF (3) to
migration time of the neutral marker
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A typical Electrophorogram
to
tr
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ANALYTICAL PARAMETERS

• Distribution Constant
• (4)
• where
• trmigration time of the species
• tomigration time of the neutral marker
• Efficiency
• (5)
• w width of the peak

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ANALYTICAL PARAMETERS
Resolution, R
(6)
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CALCULATION OF R, IONIC RADIUS

• ?e q/6??R (7)
• q/R 6??e? (8)
• where
• ? is viscosity, 8.95x10-4 N m-2s
• q z x1.6x10-19 (coulomb )
• ?e electrophoretic mobility
• R ionic radius

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Micellar Electrokinetic Capillary Chromatography
(MECC)
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Cyclodextrin Distribution Capillary
Electrochromatography (CDCE)
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Capillary Gel Electrophoresis (CGE)
Capillary is filled with a polyacrylamide gel
(C-PAGE) instead of solution as in CZE. Uniform
buffer composition. Useful for the separation of
large biomolecules based on their size
peptides, proteins, DNA and its fragments,
Oligonucleotides etc. It can also be performed
on a slab instead of capillary.
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Capillary Gel Electrophoresis (CGE)
A gel made from linear noncrosslinked
polyacrylamide (CH2CH-CO-NH2) polymer(C-PAGE) or
crosslinked polyacrylamide-bisacrylamide polymer
in a capillary is employed. Other polymers such
as dextran, agarose, and poly(ethyleneoxide) are
also used. Slab electrophoresis allows only
small voltages to be applied while the use of
capillary allows large voltages to be applied and
separations can be conducted more rapidly. The
efficiency of separation of CGE is orders of
magnitude higher than slab electrophoresis.
However the capacity of slab electrophoresis is
much higher than CGE.
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How it works?
It works by a combination of differential
migration based on charge and size and size
exclusion due to the presence of pores in the
gels. In generalsmall molecules elute faster
than larger molecules andthe predominant
mechanism of separation is size exclusion. In
CGE the capillary wall is Silanized to eliminate
electroosmotic flow. CGE is mostly used for the
separation of SDS proteins and DNA
fragments. SDS (Sodium dodecyl sulfate) SDS
associates with polypeptide fragments of
proteins obtained by the addition of a reducing
agent. The polypeptides associate with SDS to
provide molecules with different sizes but
similar charge to size ratios. They cannot be
separated by CZE and only by CGE.
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DNA Separation
DNA molecules and their fragments align
themselves in the direction of the electric
field in CGE. They do not move as a random coil
(helical structure) but in a snake-like motion
termed repatation. This makes them move faster
than that would be predicted by their size. For
larger DNA molecules and fragments the
differences in mobility get very small at
electric high fields. Often an electric field
gradient is employed to separate these molecules
to optimize their mobility differences. Pulsed
electric field is also employed in which the
magnitude and direction of the electric field are
periodically changed which amplifies size
dependent mobility.
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Capillary Isoelectric Focusing (CIEF)
This type of capillary electrophoresis is usually
used for separating proteins based on their
isoelectric points. A pH gradient is usually
employed. The isoelectric point of an amino acid
is the pH at which it exists only as its
zwitterion. NH2CH2COOH ? NH3CH2COO-
(zwitterion) pHI ½(pK1 pK2) pK1
-COOH pK2 - NH3 There is no net electrical
charge on the molecule and it behaves like a
neutral molecule. It will move along with
electroosmotic flow. When electroosmotic flow is
eliminated, it will not move at all. At pH lt
pHI, it exists as a cation. At pH gt pHI,
it exists as an anion.
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Capillary Isoelectric Focusing (CIEF)
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Capillary Isotachophoresis (CITP)
CITP is useful for the separation of either
anions or cations but not both like CZE.
Electroosmotic flow is again eliminated as in
the case of CIEF. For the separation of anions
the detector is at the anode end in contrast to
the cathode end as in CZE. A buffer with a fast
moving anion such as Cl- is placed in the
anode vial (destination vial) and a buffer with a
slow moving Anion such as heptanoate is placed in
the cathode vial (source vial). The anions of the
sample are sandwiched between the Fast moving
chloride and slow moving heptanoate anions. They
again separate into zones based on their
mobilities.
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Capillary Isotachophoresis (CITP)
When euqilibrium is established, all the anions
have The same velocity which is the velocity of
the leading anions. Hence, the name is
isotachophoresis. Since, ?
?eE The anions develop into zones with different
electric fields E and electrophoretic mobilities
?e. The Current is maintained constant for CITP
separations.
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DNA sequencing by the Sanger method
Developed by Frederick Sanger, who shared the
1980 Nobel Prize in Chemistry. In this method,
special enzymes are used to breakdown the DNA
into its fragments that terminate when a selected
base appears in the stretch of DNA being
sequenced. These fragments are then sorted
according to size by placing them in a slab of
polymeric gel and applying an electric
field. Because DNA carries negative charge, the
fragments move across the gel toward the positive
electrode. The shorter the fragment, the faster
it moves. Typically, each of the terminating
bases within the collection of fragments is
tagged with a radioactive or fluorescing probe
for identification.
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DNA sequencing example
Problem Statement Consider the following DNA
sequence (from firefly luciferase). Draw the
sequencing gel pattern that forms as a result of
sequencing the following template DNA with
ddNTP as the capper. atgaccatgattacg... Sol
ution Given DNA template
5'-atgaccatgattacg...-3' DNA synthesized
3'-tactggtactaatgc...-5'

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DNA sequencing example
Given DNA template 5'-atgaccatgattacg...-3'
DNA synthesized 3'-tactggtactaatgc...-
5' Gel pattern
http//users.rcn.com/jkimball.ma.ultranet/BiologyP
ages/D/DNAsequencing.html
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A sequencing gel
This picture is the gel false color images from
an automated DNA sequencer.
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Reading a sequencing gel
Begin at the right, which are the smallest DNA
fragments. The sequence that you read will be in
the 5'-3' direction. This sequence will be
exactly the same as the RNA that would be
generated to encode a protein. The difference
is that the T bases in DNA will be replaced by U
residues. As an example, in the problem given,
the smallest DNA fragment on the sequencing gel
is in the C lane, so the first base is a C. The
next largest band is in the G lane, so the DNA
fragment of length 2 ends in G. Therefore the
sequence of the first two bases is CG. The
sequence of the first 30 or so bases of the DNA
are CGTAATCATGGTCATATGAAGCTGGGCCGGGCCGTGC....
When this is made as RNA, its sequence would
be CGUAAUCATGGUCAUAUGAAGCUGGGCCGGGCCGUGC....
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Automated procedure for DNA sequencing
The computer scans vertically through each lane
of the gel file, and converts the pattern of
bands to an individual chromatogram with a series
of "peaks" corresponding to the DNA sequence A C
G T.  DNA migration slows over the course of
the electrophoresis, and multiple bases towards
the end may appear as a single broad band instead
of discrete "peaks".
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High-throughput seqeuncing Capillary
electrophoresis
The human genome project has spurred an effort
to develop faster, higher throughput, and less
expensive technologies for DNA sequencing.
Capillary electrophoresis (CE) separation has
many advantages over slab gel separations. CE
separations are faster and are capable of
producing greater resolution. CE instruments can
use tens and even hundreds of capillaries
simultaneously. The figure show a simple CE setup
where the fluorescently-labeled DNA is detected
as it exits the capillary.
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Sieving matrix for CE
It is not easy to analyze DNA in capillaries
filled only with buffer. That is because DNA
fragments of different lengths have the same
charge to mass ratio. To separate DNA fragments
of different sizes the capillary needs to be
filled with sieving matrix, such as linear
polyacrylamide (acrylamide polymerized without
bis-acrylamide).This material is not rigid like a
cross-linked gel but looks much like glycerol.
With a little bit of effort it can be pumped in
and out of the capillaries. To simulate the
separation characteristics of an agarose gel one
can use hydroxyethylcellulose. It is not much
more viscous then water and can easily be pumped
into the capilliaries.
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Fluorescent end labeling of DNA