Capillary

Electrophoresis

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 CONTENT
1. Introduction
S
2. Electrophoresis Overview
3. Importance Of separation technique
4. Why capillary Electrophoresis
5. What is CE
6. Types of CE
7. CE The Basics of the instrumentation
8. Theory of Capillary Electrophoresis
9. Electroosmotic Flow
10.Electroosmotic Mobility
11.Flow in CE
12.The Electrophoregram
13.Equipment of CE
14.Methods For Improving Efficiency of CE
15.Application
16.Summary and conclusion
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17.
PRINCIPLE AND INSTRUMENTATION
OF CAPILLARY ELECTROPHORESIS

PRESENTED BY:
Caspe, Nerlizabel Rose
Gammaru, Anabel A.
Bs bilogy 3-1d

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 Definition of terms
• CE- capillary electrophoresis
• E- electric field strength
• EOF- electroosmotic
• EPF-Electrophoretic flow
• Ld- length of the capillary to the detector
• Lt- total capillary length
• Uep- electrophoretic mobility
• pI- isolectric point
• V-volt
• V- voltage
• VeO- electroosmotic flow velocity
• Vep- electrophoretic velocity
 Electrophoresis—An
Overview
• Definition: The differential movement
for migration of ions by attraction
or repulsion in an electric field.
– Separation of components of a
mixture using an electric field
• v=Eq/f
– v = velocity of molecule
– E = electric field
– q = net charge of molecule
– f = friction coefficient

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 Electrophoresis- overview
cont.
• Can determine the size, shape, and
charge of a molecule
• Different forms of electrophoresis
are used for each of these factors
independently or in combination.

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 Types of Electrophoresis
• Capillary
• Native Polyacrylimide Gel
Electrophoresis (PAGE)
• Slab
• Paper
•

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 Basics
cont.
• A photocathode is then
used to measure the
absorbencies of the
molecules as they pass
through the solution
• The absorbencies are
analyzed by a
computer and they are
represented graphically

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 Capillary Electrophoresis – The
Basics Of Instrumentation
• Electrophoresis in a buffer filled, narrow-
bore capillaries
• Each capillary is about 25-100 μm in
internal diameter
• When a voltage is applied to the solution,
the molecules move through the solution
towards the electrode of opposite charge
• Depending on the charge, the molecules
move through at different speeds
– Separation is achieved
•
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10
Cont….

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Capillary Electrophoresis
Apparatus

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 Electrophoresis
• a separation technique based on
 the differential transportation of
charged species in an electric field
through a conductive medium.
• Primary candidates for CE separation
are ions.
• The basic instrumental set-up, consists
of a high voltage power supply (0 to
30 kV), a fused silica (SiO2) capillary,
two buffer reservoirs, two electrodes,
and an on-column detector.
 Capillary electrophoresis
(CE)
• is a family of related techniques that employ
narrow-bore (20-200 mm i.d.) capillaries to
perform high efficiency separations of both
large and small molecules.
• separations are facilitated by the use of
high voltages, which may generate
electroosmotic and electrophoretic flow
of buffer solutions and ionic species,
respectively, within the capillary.
• The properties of the separation and the
ensuing electropherogram have
characteristics resembling a cross
between traditional polyacrylamide gel
electrophoresis (PAGE) and modern high
 Electrophoresis
terminology
• under ideal conditions, nothing is
retained, so the analogous term
becomes migration time.
• The migration time (tm) is the
time it takes a solute to move
from the beginning of the capillary
to the detector window.
• Other fundamental terms are defined
below. These include the
electrophoretic mobility, mep
(cm2/Vs), the electrophoretic
velocity, vep (cm/s), and the
The relationships between these factors are shown in Equation 1.

Equation 1 is only useful for determining the

apparent mobility. To calculate the actual
mobility, the phenomenon of electroosmotic flow
must be accounted for. To perform reproducible
electrophoresis, the electroosmotic flow must be
carefully controlled.
 Electroosmosis
• This phenomenon is a consequence
of the surface charge on the wall of
the capillary.
• A vitally important feature of CE is
the bulk flow of liquid through the
capillary.
• This is called the electroosmotic flow
and is caused as follows
The negatively-charged wall attracts positively-charged ions
from the buffer, creating an electrical double layer. When a
voltage is applied across the capillary, cations in the diffuse
portion of the double layer migrate in the direction of the
cathode, carrying water with them.
The result is a net flow of buffer solution in the direction of
the negative electrode.
Stern ’ s model of the double - layer charge distribution at a
negatively charged capillary wall leading to the generation 19
of a zeta potential and EOF
 Electroosmotic Mobility


• Zeta Potential
• The change
in potential
across a
double
layer
• Proportional
to the
charge on
the capillary
walls and to
the 20
Effects of pH
 Flow Profile in CE
• A further key feature of EOF is that it has flat flow
profile, which is shown in Figure alongside the
parabolic flow profile generated by an external
pump, as used for HPLC.
•
• EOF has a flat profile because its driving force (ie.,
charge on the capillary wall) is uniformly
distributed along the capillary, which means that
no pressure drops are encountered and the flow
velocity is uniform across the
 capillary.


• In HPLC, in which frictional forces at the column walls

cause a pressure drop across the column, yielding
a parabolic or laminar flow profile.
• The flat profile of EOF is important because it
minimizes zone broadening, leading to high
separation efficiencies that allow separations on
the basis of mobility differences as small as 0.05
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 The
Electropherogram
• The data output from CE is
presented in the form of
an electropherogram,
which is analogous to a
chromatogram.
• An electropherogram is a
plot of migration time vs.
detector response.
• The detector response is
usually concentration
dependent, such as UV-
visible absorbance or
fluorescence.
• The appearance of a typical
electropherogram is
shown in Figure for the
separation of a
threecomponentmixture of 24
cationic, neutral and
 Equipment
• Capillary tube
• Varied length
but
normally
25-50 cm
• Small bore
and
thickness of
the silica
play a role
– Using a
smaller
internal
diamet 25
 Equipment Cont….

• Detector
– UV/Visible absorption
– Fluorescence
– Radiometric (for radioactive
substances)
– Mass Spec.

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 Effects of voltage and
temperature
• Both the electroosmotic and
electrophoretic velocities are directly
proportional to the field strength, so the
use of the highest voltages possible will
result in the shortest times for the
separation.
• Short separation times will give the
highest efficiencies since diffusion is the
most important feature contributing to
bandbroadening.
• The limiting factor here is Joule heating.
Experimentally, the optimal voltage is
determined by performing runs at
• The electrophoreticmobility and the
electroosmoticflow expressions both
contain a viscosity term in the
denominator.
• Viscosity is a function of temperature;
therefore, precise temperature control
is important. As the temperature
increases, the viscosity decreases;
thus, the electrophoretic mobility
increases as well.
• Some buffers such as Tris are known to
be pH-sensitive with temperature. For
complex separations such as peptide
maps, even small pH shifts can alter
HINTS FOR IMPROVING EFFICIENCY
OF CE
 Buffers
• Additives for CZE
• Additives for HPCE
• Hints
• Ionic Strength in HPCE
• PKa Values of Common Buffers
• Proteins, Choosing a Proper Buffer
 
 Capillaries
• Conditioning
• Dimensions, Changing
• How to Properly Cut A Capillary (Animated)
• Storage
 
• Data Analysis
• Migrating Peak Correction
 

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 Applications
• Advantages
• Fast
• Analysis of • Small Sample
carbohydrates • Relatively
inexpensive
• Analysis of inorganic • Automated
anions/metal ions 

• DNA profiling • Disadvantages

• Protein identification • Cannot identify
neutral
 species
• Joule Heating
• Cannot discern
shape

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 Summary


1. CE is based on the principles of

electrophoresis.
2. The speed of movement or migration of
solutes in CE is determined by their
3. size and charge. Small, highly charged
solutes will migrate more quickly than
large, less charged solutes.
4. Bulk movement of solutes is caused by EOF.
5. The speed of EOF can be adjusted by
changing the buffer pH used.
6. The flow profile of EOF is flat, yielding high
separation efficiencies.
7. The data output from CE is called an
electropherogram.
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 Conclusion
• It is the most efficient separation
technique available for the analysis
of both large and small molecules.
• DNA Profiling, protein identification,
 inorganic metals and ions can be
detected easily by this method.
•

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 Modes of capillary
electrophoresis
• Capillary zone electrophoresis
• Isoelectric focusing
• Capillary gel electrophoresis
• Isotachophoresis
• Micellar electrokinetic capillary
chromatography
 Capillary zone
electrophoresis
• Simplest form of CE.
– Capillary zone electrophoresis (CZE),
(free solution capillary
electrophoresis)
• The separation mechanism is based
on differences in the charge-to-
mass ratio
• Fundamental to CZE are
homogeneity of the buffer
solution and constant field
• The net charge is usually pH
dependent.
• Separations of both large and small
molecules can be accomplished by
CZE.
• Even small molecules, where the
charge-to-mass ratio differences
may not be great, may still be
separable.
 Capillaries
• Capillaries with an internal diameter
of 25-75 mm are usually employed.
– Fused silica is the material of choice
due to its UV transparency,
durability (when polyimide coated),
and zeta potential.
 Effect of pH.
• At a high pH where EOF is substantial, the
order of migration will be cations, neutrals,
and anions. None of the neutral molecules will
be separated since the net charge is zero.
• The anions will still migrate toward the cathode
because the EOF is greater than the
electrophoretic migration.
• At lower pH where the EOF is greatly reduced,
both cations and anions can still be
measured, although not in a single run. To
measure anions, the anode must be beyond
the detector window. Likewise, to measure
cations, the cathode must reside beyond the
detector window.
• The proper electricalconfiguration is achieved
by simply reversing the polarity of the
 Buffers
• A wide variety of buffers can be
employed in CZE.
• buffer is most effective within one or
two pH units of its pI.
• Zwitterionic buffers such as bicine,
tricine, CAPS, MES, and Tris are also
common, particularly for protein and
peptide separations. The advantage
of the zwitterionic buffer is its low
conductivity when the buffer is
employed around the pI.
• The advantage therein is the low
current draw andthus reduced Joule
 Isoelectric focusing
• The fundamental premise of isoelectric
focusing (IEF) is that a molecule will
migrate so long as it is charged.
• IEF is run in a pH gradient where the pH is
low at the anode and high at the cathode .
• The pH gradient is generated with a series
of zwitterionic chemicals known as
carrier
• ampholytes.
• When a voltage is applied, the
ampholyte mixture separates in the
capillary.
– Ampholytes that are positively charged will
migrate towards the cathode while those
negatively charged migrate towards the
 Application
• In addition to performing high
resolution separations, IEF is
useful for determining the pI of a
protein.
• IEF is particularly useful
forseparating immunoglobulins,
hemoglobin variants and post-
translational modifications of
recombinant proteins.
 Capillary gel
electrophoresis
• Traditional gel electrophoresis is
conducted in an anticonvective
medium such as polyacrylamide or
agarose.
• The composition of the media can
also serve as a molecular sieve to
perform size separations
•
 There are two fundamental
classes of gels
• physical gel
• Chemical gels
•
 Isotachophoresis
• was the most widely used
instrumental capillary
electrophoretic technique, although
the capillaries were quite wide
(250-500 mm) by today’s
standards.
 Micellar electrokinetic
capillary chromatography
• Micelles are amphiphilic aggregates
of molecules known as
surfactants.
• They are long chain molecules
(10-50 carbon units) and are
characterized as possessing a long
hydrophobic tail and a hydrophilic
head group.
 Thank you !!!
Caspe, Nerlizabel Rose C.
Gammaru, Anabel A.
BS. Biology 3-1D