Capillary

Electrophoresis

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1. Introduction
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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PRINCIPLE AND INSTRUMENTATION
OF CAPILLARY ELECTROPHORESIS

PRESENTED BY:
Caspe, Nerlizabel Rose
Gammaru, Anabel A.
Bs biology 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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Cont….

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11
Capillary Electrophoresis
Apparatus

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 Electrophoresis
terminology
• 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 electric
field strength, E (V/cm).
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
• 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 of17a
 Electroosmotic Mobility

• Zeta Potential
• The change in
potential across a
double layer
• Proportional to the
charge on the
capillary walls and
to the thickness of
the double layer.
– Both pH and ion
strength affect
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the mobility
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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21
 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 threecomponent
mixture of cationic, neutral
and anionic solutes. 22
 Equipment
• Capillary tube
• Varied length but
normally 25-50 cm
• Small bore and
thickness of the silica
play a role
– Using a smaller
internal diameter and
thicker walls help
prevent Joule Heating,
heating due to voltage

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 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
increasing voltages until deterioration in resolution is
noted.
• The electrophoretic mobility and the electroosmotic
flow 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
the selectivity.
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
• Relatively inexpensive
carbohydrates
• Automated
• Analysis of inorganic
anions/metal ions • Disadvantages
• DNA profiling • Cannot identify
neutral species
• Protein identification
• 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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Thank you !!!
Caspe, Nerlizabel Rose C.
Gammaru, Anabel A.
BS. Biology 3-1D