Electrophoresis-Part II

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 Paper Electrophoresis
• The technique of paper electrophoresis is simple
and inexpensive and requires only micro
quantities of plasma for separation.
• The support medium is a filter paper
• The electrophoresis apparatus in its simplest form
consists of two troughs to contain buffer solution,
through which electric current is passed.
• Frequently used in isolating proteins, amino acids
and oligopeptides.
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 Principle
“The charge carried by a molecule depends on
the pH of the medium. Electrophoresis at low
voltage is not usually to separate low
molecular weight compounds because of dif
fusion, but it is easier to illustrate the
relationship between charge and pH with amino
acids than with proteins (or) other
macromolecules”.

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 APPARATUS
• The equipment required for electrophoresis
consists basically of two items, a POWER PACK
and an ELECTROPHORETIC CELL.
• Power pack :provides a stabilized direct current
and has controls for both voltage & current
output, which have an output of 0 to 500V and 0
to 150mA are available.
• The Electrophoretic cell: contains the electrodes,
buffer reservoirs, a support for paper and a
supporting transparent insulating cover.
• The electrodes are usually made of platinum.
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• The two arrangements of the
filter strips are commonly
used.
The horizontal & vertical
arrangements.
Both the arrangements are
equally
viable & the choice usually
depends
upon personal preferences.
• Filter paper:
• Paper of good quality should
contain at least 95% α–
cellulose and should have
only a very slight adsorption
capacity.
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 PROCEDURE

1) A long strip of filter paper is moistened with a suitable buffer solution

of the desired pH and the sample is applied transversely across the
central part of the strip.
2) Ends are fixed to dip in buffer solutions in two troughs fitted with
electrodes.
3)Electric field of about 20 volts/cm is established.
4)The charged particles of sample migrate along the strip towards
respective electrodes of opposite polarity, according to net charges,
sizes and interactions with the solid matrix.
5)Homogeneous group of particles migrate as a separate band
6)The electrophoresis is carried out for 16-18 hours.
7) Separated Proteins are fixed to a solid support using a fixative such as
Acetone or Methanol
8)Proteins are stained (bromophenol blue) to make them visible
9) The separated proteins appear as distinct bands 7
 OBSERVATION
• The different fractions appear
as blue colored bands across
the filter paper starting from
the moving boundary
backwards.
• If a quantitative estimation is
required for each fraction, the
bands may be carefully cut
and eluted, or the bands may
be scanned optically in a
densitometer.
• In human plasma five
different bands can be
identified on paper
electrophoresis
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 APPLICATIONS
• Serum analysis for diagnostic purpose is routinely carried about by paper
electrophoresis.
• Muscle proteins, egg white proteins, milk proteins & snake, insect venom
analysis done by this technique

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Gel Electrophoresis

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• Gel electrophoresis is a process that separates
fragments of DNA based on their sizes by using
electricity to carry them through a gel.
• Separation is brought about through molecular
sieving technique, based on the molecular size of
the substances. Gel material acts as a "molecular
sieve”.
• Gel is a colloid in a solid form (99% is water).
• It is important that the support media is electrically
neutral.
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• The larger the fragments whether DNA,RNA or
protien, the slower it will move through the gel
matrix.
• DNA can be separated by fragment size,
smallest fragment traveling largest distance and
larger fragment traveling shortest distance.
• Different types of gels which can be used are;
Agar and Agarose gel, Starch, Sephadex,
Polyacrylamide gels.
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• Agar gel is used for separation of different
types of protein mixtures as well as nucleic
acids.
• Polyacrylamide is most suitable for separation
of nucleic acids.
• It is also frequently used in separating
proteins, peptides and amino acids from
microgram quantities of mixed samples.

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 PRINCIPLE
• Any charged ion or molecule migrates when
placed in an electric field, the rate of
migration depend upon its net charge, size,
shape and the applied electric current.

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 PROCEDURE
Step1.Place DNA into tubes :DNA can come from tissue or
body fluid, such as cheek cells, blood, skin, and hair.
Step 2.Polymerase Chain Reaction: The polymerase chain
reaction uses a machine called a thermo cycler to
quickly copy a piece of DNA. Continue these three steps
30 to 40 times to get lots of copies of DNA.
Step 3.Place restriction enzymes into DNA: The restriction
enzymes cut the DNA into different sizes according to
it’s sequence.
Step 4.Dye DNA and place into gel : The gel is made out of
wells at one end so that DNA can be loaded into the gel.
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Step 5: Run electric current through gel : DNA is
negatively charged so it will move towards the
positively charged end of the gel.
Step 6: Smaller pieces of DNA travel farther
than Larger pieces of DNA

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 APPLICATION
• Solve criminal cases
• Solve paternity cases
• Diagnose genetic diseases
• Determine genetic kinship among species

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 ADVANTAGES
• Easy to do
• DNA does not get ruined in the process
• You only need a small amount of DNA to start
with
• DNA can be detected no matter what size it is
DISADVANTAGES
• Expensive
• Time consuming process
• Uses hazardous material
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 CAPILLARY ELECTROPHORESIS
• The principle behind electrophoresis is that charged molecules will migrate toward
the opposite pole and separate from each other based on physical characteristics.
• Capillary electrophoresis has grown to become a collection of a range of separation
techniques which involve the application of high voltages across buffer filled
capillaries to achieve separations .
• Capillary electrophoresis, then, is the technique of performing electrophoresis in
buffer filled, narrow-bore capillaries, normally from 25 to 100 mm in internal
diameter (ID).
• A high voltage (typically 10-30 kV) is applied.
• Capillaries are typically of 50 μm inner diameter and 0.5 to 1 m in length.
• Due to electro osmotic flow, all sample components migrate towards the negative
electrode.
• The capillary can also be filled with a gel, which eliminates the electro osmotic flow.
Separation is accomplished as in conventional gel electrophoresis but the capillary
allows higher resolution, greater sensitivity, and on-line detection.
• The capillary is filled with electrolyte solution which conducts current through the
inside of the capillary. The ends of the capillary are dipped into reservoirs filled with
the electrolyte.
• Electrodes (platinum) are inserted into the electrolyte reservoirs to complete the19
Sample application is done by either
a)High voltage injection-potential is applied causing the sample to enter
capillary by
combination of ionic attraction and electroosmotic flow.
b)Pressure injection-pressure difference is used to drive the sample into
capillary by applying vaccum.
• When PD is applied net migration occurs in the direction of cathode.
• Even substance with net negative charge migrate in the direction of cathode
due to the phenomenon called as Electro Osmotic Flow.
• Neutral molecule moves at the same speed as the EOF. Positively charged
species move faster, speed is sum of EOF and Electrophoretic mobility.
Negatively charged molecules lag behind. 20
 ELECTROOSMOTIC FLOW
• The surface of the silicate glass capillary contains negatively-charged
functional groups that attract positively-charged counter ions.
• The positively-charged ions migrate towards the negative electrode and
carry solvent molecules in the same direction.
• This overall solvent movement is called electro osmotic flow.
• During a separation, uncharged molecules move at the same velocity as
the electro osmotic flow (with very little separation).
• Positively-charged ions move faster and negatively-charged ions move
slower.

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• A small volume of sample is moved into one end of the capillary.
• The capillary passes through a detector, usually a UV absorbance detector,
at the opposite end of the capillary.
• Application of a voltage causes movement of sample ions towards their
appropriate electrode usually passing through the detector.
• A plot of detector response with time is generated which is termed an
electropherogram

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Different modes of Capillary electrophoresis
The different modes of capillary electrophoresis are
1. Capillary zone electrophoresis (CZE)
2. Capillary gel electrophoresis (CGE)
3. Micellar electro kinetic capillary chromatography
(MEKC)
4. Capillary electro chromatography (CEC)
5. Capillary iso-electric focusing (CIEF)
6. Capillary isotachophoresis (CITP).
7. Electro chromatography (EKC)
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• The capillary electrophoresis can be classified into continuous
and discontinuous systems as shown in Figure.
• A continuous system has a background electrolyte acting
throughout the capillary as a buffer. Continuous system is
further classified into kinetic (constant electrolyte
composition) and steady-state (varying electrolyte
composition) processes.
• A discontinuous system keeps the sample in distinct zones
separated by two different electrolytes.

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1. Capillary Zone Electrophoresis (CZE)
• Capillary Zone Electrophoresis (CZE), is the most commonly used
technique. It is also known as free solution capillary
electrophoresis. The separation is based on the differences in
electrophoretic mobility. The velocity of the ion is directly
proportional to the electrophoretic mobility and the magnitude
of the electric field. The electrophoretic mobility is directly
proportional to the charge on the molecule and inversely
proportional to the viscosity of the solvent and radius of the
atom.
2. Capillary Gel Electrophoresis (CGE)
• A gel matrix is used in the capillary. Separation is based on the
size of the components. Different sized but same mobility
components are separated by this technique.CGE is used for
protein and DNA separation. Smaller molecules move faster and
the bigger molecules are moving slowly in CGE. 25
3. Micellar electro kinetic capillary chromatography
(MEKC)
• Solutes are partitioning between micelles and
the solvent in MEKC. When a surfactant is added to a
solution, aggregates of surfactant molecules are
formed above the critical micelle concentration. These
aggregates are called as micelles. The aggregates have
polar negatively charged surfaces outside (Hydrophilic)
and non polar surface inside the micelle(hydrophobic).
• Hydrophobic molecules spend more time with
micelles, while the hydrophilic molecule migrates at a
faster rate with the solvent.
Factors that affect the electro osmotic flow in MEKC are pH,
surfactant concentration, additives, and polymer coatings of
the capillary wall.
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4. Capillary electro chromatography (CEC)
• The capillary is packed with silica
particles. The separation principle is same as
column chromatography. Electroosmotic flow
is present due to the charges on the silica. The
flow obtained is a plug type flow.

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• 5. Capillary iso-electric focusing (CIEF)
• CIEF is commonly used to separate peptides and proteins. These
molecules are zwitterionic in nature because they contain both positive
and negative charges. Functional groups attached to the main chain and
the pH of the environment determines the charge on the molecule. Each
molecule has a specific iso electric point (pI). When the surrounding pH
is equal to this pI, the molecule carries no net charge (zwitter ion form).
• At pH lower than pI – molecule exist as positive ion
• pH higher than pI – molecule exist as negative ion.
• Since the charge on the molecule changes with pH, a pH gradient can be
used to separate molecules in a mixture. A pH gradient is maintained
across the capillary and a voltage is applied. The anode (+ve voltage) of
the capillary sits in acidic solution (low pH), while the cathode (-ve
voltage) sits in basic solution (high pH). The components of the mixture
migrate at different rates to the pH value that equals the pI value of each
component. At pI the molecule exists as zwitterion and its migration is
stopped.
• Pressure is applied to move the complete pH gradient through the
capillary and the components pass through the detection window. 28
6. Capillary isotachophoresis (CITP).
• It is a discontinuous system, based on principle of moving boundary
electrophoresis. The technique of isotachophoresis depends on the
development of potential gradient.
• A leading electrolyte (e.g. chloride) with a higher mobility than the analytes,
and a trailing
• electrolyte (e.g. glycinate) with a lower mobility is used.Solution in which the
separation takes place is normally an aqueous medium, which contains sucrose
toprovide a higher density to the solution.
• The technique of Isotachophoresis depends on the development of a potential
gradient.
• The analytes are positioned between the electrolytes and, when the voltage is
applied, they migrate in order of decreasing mobility. This establishes the
potential gradient and from that point on, all the analytes move at the same
speed.
• Individual zones border one another but represent completely separated
components without overlap.
• In isotachophoresis no buffer is mixed with the sample, so current flow is
carriedonly by charged sample ions.
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7. Electro chromatography (EKC)
• It is used in the separation of chiral compounds by
differential interaction of enantiomers with cyclodextrins.
• Advantages of CE
• 1. High separation efficiency
• 2. Rapid technique and high resolution of separation
• 3. Detection is simple.
• 4. Easy operation and low waste generation,
• Disadvantages
• 1. The small diameter of the capillary causes dissipation of heat,
leading to increased diffusion.
• 2. Sample sticking to capillary walls
• 3. Inconsistent retention times.
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Applications
• 1.DNA finger printing: Using CE, individual nucleotides can be
identified.
• 2. Drug analysis: Drugs and related compounds can be identified
with CE
• 3. To study the characteristics of protein: Proteins are
amphoteric in nature and they can
• be separated and purified by CE using the iso electric point pI.
• 4. DNA sequencing: DNA sequencing is the process of
determining the order of nucleotides in DNA. This sequencing
can be carried out with CE
• 5. Capillary electrophoresis is also used for the simultaneous
estimation ofNH4+,Na+,K+,Mg2+ and ca2+ in biological fluids. 31