Lecture note : CE ??? ?????

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Dong-Sun Lee/ CAT-Lab / SWU
Chapter 33 Capillary Electrophoresis
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History Electrophoresis as an analytical tool was
introduced by the Swedish chemist Arne Tiselius,
first in his doctoral thesis in 1930. For his
pioneer work in this field, Tiselius was awarded
the Nobel prize in 1948. The first appearance of
capillary electrophoresis can be found in 1981
with the publication of an article by J.W.
Jorgenson and K.D. Lukacs, working at the
University of North Carolina, in Analytical
Chemistry, 53 (1981) 1298.
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Capillary Electrophoresis Capillary
Electrophoresis (CE) is a technique that is
utilized to separate complex mixtures of
biological and chemical species. The technique
employs capillary columns, buffers (with or
without additives), and high voltages to perform
high resolution separations based on size, shape
and charge to mass ratio of organic and inorganic
molecules. Both qualitative and quantitative
separations are achieved with a number of modes
of CE, including free-zone CE, capillary gel
electrophoresis, micellar electrokinetic
capillary chromatography and isoelectric
focusing. A number of detectors are utilized
including UV-visible diode array, fluorescence,
mass spectrometry, and indirect amperometric.
Examples Main component drug assay of
pharmaceuticals. Identification of
impurities in pharmaceutical and agricultural
products. Chiral separations.
Separation of DNA and DNA fragments.
Separation of nucleotides, nucleosides and bases.
Separation of proteins and peptides.
Characterization of amine functional and charged
polymers.
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Capillary electrophoresis (CE) is a technique in
which an electrophoretic separation takes place
in a narrow-bore fused silica capillary. The
capillaries typically used in CE are commercially
available at reasonable cost (about 4/meter.)
We use capillaries that range from 30 to 50
centimeters in length, 0.150 to 0.375 millimeters
in outer diameter, and have a 0.010 to 0.075
millimeter diameter bore. In a CE separation,
the capillary is filled with buffer and each end
is immersed in a vial of the same buffer. A
sample of analyte is injected at one end, either
by electrokinesis or by pressure, and a electric
field of 100 to 700 volts/centimeter is applied
across the capillary. As the analyte mixture
migrates through the capillary due to the applied
electric field (electrophoresis), differing
electrophoretic mobilites drive each of the
components into discrete bands. At the other end
of the capillary each of the separated analytes
is detected and quantified. Electrophoretic
mobility is proportional to the charge of the
molecule divided by its frictional coefficient.
This is approximately equal to the charge to mass
ratio of the molecule. So in general any
molecules with differering charge to mass ratios
can be separated by CE. The diagram below is an
attempt to help demonstrate this.
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Basic principles of CE The electrophoretic
separations of ions The mechanism of separation
in electrophoresis is based on the migration of
charged particles in an applied electric field.
Different particles with different charges and
/or sizes migrate with different velocities. The
electrostatic force F exerted on an ion i in
solution is proportional to the net charge of the
ion (qi) and the electric field strength(E, in
V/cm) F qi E The direction of
the force is to the electrode with a charge
opposite to that of the ion. Under the influence
of the electrostatic force the charged ion is
accelerated and starts migrating. Its movement is
then opposed by viscosity(?) of the solution,
which increase proportional with the velocity
(vi, in cm s1) of the ion. For a spherical
particle(radius, ri) the viscosity is given by
the Stokes equation F 6? ?
rivi After a very short acceleration time the
opposing force (electrostatic and viscous) cancel
each other out and the particle then moves with
constant velocity through the solution.
vi (qi E) / (6? ? ri) The electrophoretic
mobility (?i) has been defined as ? i
vi / E qi / (6? ? ri)
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?
Anode
Cathode

?
?


Movement of charged particles under the influence
of an applied electric filed
Electric force (qE)
Viscous drag
Cathode
Viscous drag
Anode
?q
q
?

Electric force (qE)
E
Forces acting on charged particles moving in an
electric field.
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The net effect for the mobility of an ion 1.
Mobility is directly proportional to the charge
of an ion 2. Mobility is inversely proportional
to the viscosity of the solvent 3. Mobility is
inversely proportional to the radius of particle
(represented by the diffusion
coefficient) It is clear that different ions can
be separated when they differ either in charge or
radius or, better, when their charge/size ratio
differs. Wim Kok, Capillary Electrophoresis
Instrumentation and Operation, Chromatographia,
Suppl. 51, S9, 2000. Patrick Camilleri, Capillary
Electrophoresis-Theory and Practice, CRC, 1993,
p66-67
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Factors affecting electrophoretic mobilities 1.
Nature of the charged particles net charge,

size,
relative mass,

charge-to-size ratio 2. Nature of the
electrophoretic system 1) The ionic
composition of the electrophoresis buffer
2) The temperature 3) The pH of the
electrophoresis buffer 4) The applied
voltage 5) The type of support medium
pore size
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Principles of electroosmosis When an aqueous
solution of electrolytes, as used in
electrophoresis, is contact with the wall of the
separation capillary, there is a charge
separation between the wall and the solution.
This can be caused by ionization of the wall
material or by specific adsorption of ions from
the solution to the wall. With fused silica
capillaries the wall is usually negatively
charged. Free silanol groups (which has a pKa of
6 to 7) on the surface of the fused silica are
deprotonated(at pHgt 1.5), leaving negative Si-O
groups. Since the system as a whole must be
electrically neutral, the solution in the
separation compartment has a net positive charge.
This excess of positive ions is located in the
solution close to the wall, due to the
electrostatic attraction by the negative
wall. When a voltage is applied between the ends
of the capillary, the electric field exerts a
force on the excess of positive charge in the
solution close to to the wall. This force drives
the solution in the capillary as a whole in the
direction of the negative electrode. A constant
flow of the solution results when the viscous
forces in the thin layer of solution near the
wall counteract the electrostatic force. This
phenomenon is called electro-osmosis or
electro-endoosmosis.
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The principle of electroosmosis.
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(a) Electric double layer created by negatively
charged silica surface and nearby cations. (b)
Predominance of cations in diffuse part of the
double layer produces net electroosmotic flow
toward the cathode when an external is applied.
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High ectroosmotic mobility
Low ectroosmotic mobility
Hydrodynamic flow profiles superimposed over the
EOF when differences in the electroosmotic
mobility exist over the length of the capillary.
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Flow profiles for liquids under (a)
electroosmotic flow and (b) pressure-induced flow
(parabolic velocity profile).
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Total electrophoretic mobilty is the vector sum
of the electrophoretic mobility of the sample and
the effective mobility due to electroosmotic flow.
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Types of electrophoretic separations 1) Classical
electrophoresis a. Moving boundary
electrophoresis b. Zone electrophoresis
paper, cellulose acetate, starch gel,
polyacrylamide gel,
agarose gel / immuno, rocket /
tube, slab, disc, i. Horizontal
ii. Vertical
c. Steady state electrophoresis
i. Isoelectric focusing ii.
Isotachophoresis 2) Capillary zone
electrophoresis (CZE) 3) Capillary
isotachophoresis (CITP) 4) Capillary gel
electrophoresis (CGE) 5) Capillary isoelectric
focusing (CIEF) 6) Micellar electrokinetic
capillary chromatography (MECC) 7) Capillary
electrochromatography (CEC)
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Initial
Sample ABC
Leading electrolyte
Final
A
AC
ABC
Moving boundary electrophoresis.
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Initial
Buffer
Buffer
ABC
Final
A
C
B
Buffer
Buffer
Zone electrophoresis.
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Initial
TerminalBuffer
Leading Buffer
ABC
Final
TerminalBuffer
Leading Buffer
A
C
B
pH 2 4 6 8 10
Isotachophoesis
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Initial
Buffer
Buffer
ABC
pH 2 4 6 8 10
Final
B
A
C
Buffer
Buffer
pH 2 4 6 8 10
Isoelectric focusing.
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Moving boundary electrophoresis.
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Horizontal electrophoresis.
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Isoelectric focussing
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Vertical paper electrophoresis.
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Tube gel electrophoresis.
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Disc electrophoresis.
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Electrochromatography In recent years a number
of new separation techniques employing high
voltages and narrow bore capillaries have been
brought to light. These include
Capillary Electrophoresis (CE or CZE)
Capillary Gel Electrophoresis (CGE)
Micellar Electrokinetic Capillary Chromatography
(MECC) The first two techniques in the above
list can be viewed as high tech instrumental
analogues of the well know slab-gel
electrophoresis, whereas MECC makes use of a form
of chromatographic partition to achieve the
separation of the components. However, the
capillary electro- technique which most closely
resembles modern HPLC is electrochromatography.
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Instrumentation 1) Capillary 2575?m ID fused
silica capillary with a thin outer coating of
polyimide 2) Injection volume 1 ?l for a 50 cm
long, 50 ?m ID 3) Detector a. Optical
absorbance detector b. Laser based absorbance
detector c. Refractive index detection d.
Thermooptical absorbance e. Fluorescence
detector f. Chemiluminescence detector g.
Electrochemical detector Conductivity,
Amperometric h. Radioactivity detector I.
Hyphenated detection CE-MS 4) Power supply 5)
Buffer and additives
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Capillary electrophoresis instrument.
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