Transcript Lecture 16

CAPILLARY ELECTROPHORESIS
Electrophoresis is the movement of electrically
charged particles through a media caused by an
electric current.
A high voltage, up to 30 kV is applied causing positively
charged cations to migrate towards the negative electrode
(cathode) and negatively charged anions to move toward
the positive electrode (anode).
Electrophoresis and Separation
The main principle of separation is that small
molecules move faster than large molecules, and
molecules with higher charges move faster than
ones with lower charges. Therefore, molecules
are separated based on their charge/size ratios.
There is another factor in capillary
electrophoresis that contributes the movement of
ions and produces additional separation ELECTROOSMOTIC FLOW
ELECTROOSMOTIC FLOW
When an electric field is applied, a layer of
positive ions forms along the capillary walls. This
mobile layer of cations is pulled towards the
cathode. This movement of cations is great
enough to drag the whole buffer system (bulk
flow) along through the capillary tube.
ELECTROOSMOTIC FLOW
Normally, cations migrate towards the cathode,
and anions towards the anode. However, the
electroosmotic flow is great enough to move both
positive and negatively charged molecules
towards the cathode. This makes it possible to
analyze both cations and anions in a single run.
ELECTROOSMOTIC FLOW
Under normal hydrodynamic pressure, friction along the
wall of the capillaries causes the flow to slow down
compared to the flow in the center of the capillary. This
difference in flow velocities from the side wall to the center
of the capillary causes band broadening. Electroosmotic
flow is generated at the side walls of the capillary and
because of this, the flow profile is much more uniform than
in normal capillary flow.
EFFICIENCY
Plate Height, H
van Deemter Plot
H = A + B/u + (Cs + Cm) u
A + B/u + Cu
Mass Transfer (both), Cu
Multipath Term, A
Longitudinal diffusion, B/u
Linear Velocity, u
Plate Height, H
EFFICIENCY
X
H = A + B/u + (Cs + Cm) u
B/u + Cu
Mass Transfer (both), Cu
Longitudinal diffusion, B/u
Linear Velocity, u
Plate Height, H
EFFICIENCY
X
X
H = A + B/u + (Cs + Cm) u
Therefore Capillary Electrophoresis
is very efficient. Typical plate counts
(N) for capillary electrophoresis are
in the range of 100,000 – 200,000
compared to 5000 to 20,000 for
B/u
typical HPLC.
Longitudinal diffusion, B/u
Linear Velocity, u
SAMPLE INJECTION METHODS
Sample introduction is very important since
extremely small samples are used (1 – 50
nanoliter or even less). These very small sample
sizes make reproducible quantification of
analytes difficult.
There are advantages to using very small
samples. For Example: The end of the capillary
can be heated and pulled down to an extremely
small diameter. These very fine points on the
end of the capillary can be used to sample inside
single cells or even within structures of the cell.
SAMPLE INJECTION METHODS
Hydrostatic Injection:
immerse one end of the capillary in the sample
container and either pressurizing the container
or applying a vacuum to the other end of the
capillary.
Gravity Injection:
the capillary is inserted into the sample vial
and the sample vial is raised up above the level of
the column outlet and the sample is siphoned into
the column. The amount of sample introduced is
based on time the capillary is placed in the
sample.
SAMPLE INJECTION METHODS
Electrokenetic injection:
One end of the capillary is inserted into the
sample and voltage is applied across the column.
The amount of sample taken up is based on the
electrophoretic mobility of the sample ions, the
electroosmotic flow, and differences in the ionic
strength of the sample and buffer.
This method can produce biases in sample
introduction since mobile ions are taken up at a
faster rate. However, electrokenetic injection
can be used as a pre-concentration step and
significantly lower the detection limits.
Micellar Electrokinetic Capillary
Chromatography
When sodium dodecyl sulfate
(SDS) is added to the buffer above
a critical concentration, it forms
aggregates
called micelles.
One end of the SDS molecule is a
hydrophillic SO3 group while the
rest of the molecule is a
hydrophobic 12 carbon chain.
The molecules group together
with their hydrophobic ends
together and their negative
charges to the surface of the
micelle.
Micellar Electrokinetic Capillary
Chromatography
MEKC can be thought of as a
combination of electrophoresis
and reversed phase HPLC.
The hydrophobic part of the
micelle acts like the C-18
molecules in an HPLC column. A
compound of intermediate water
solubility will partition between
the buffer and the hydrophobic
parts of the micelle. Since the
micelle moves slower than
buffer, analytes that partition
mostly in the buffer, will move
relatively quickly while analyte
molecules that partition mostly
into the micellewill move more
slowly.
CHIRAL CHROMATOGRAPHY

Enantiomers can be separated with capillary
electrophoresis by the use of chiral additives to
the mobile phase. Cyclodextrins are commonly
used for this purpose. Cyclodextrins are rings of
usually 6, 7, or 8 glucose molecules. Optical
isomers associate significantly differently with
the cyclodextrins and can be separated.
Factors Affecting Separations
Voltage- Generally produces sharper peaks and shortens
analysis time (up to a point).
Capillary Internal Diameter.- Better separations are
achieved with smaller diameter. Small ID limits the amount
of sample that can be used and the detector performance
is lower.
Buffer – Increasing the buffer pH will increase EOF.
Increasing the buffer ionic strength will decrease EOF, but
in many cases it will result in increased resolution. Use of
different buffer ionic species can have a dramatic effect on
resolution.
Modifiers. Organic modifiers can be used to increase the
solubility of many analytes. Organic modifiers such as
methanol reduce flow while acetonitrile increases flow.
ADVANTAGES OF CAPILLARY
ELECTROPHORESIS
• High Efficiency (N =100,000 to 1,000,000)
• Short Analysis Times
• Simultaneous Separation of Anions and
Cations
• Small Sample Volumes (pico- to nanoliters)
• Low Consumption of Mobile Phase
• Inexpensive Columns ($5 - $100)
• Extremely Versatile