Capillary Electrophoresis and

Capillary Electrochromatography
 Electrophoresis
Electro- flow of electricity
Phoresis- to carry across

Electrophoresis is the motion of the

dispersed particles relative to a fluid under
the influence of spatially uniform electric
field.
This electro-kinetic phenomenon was
observed in 1807 by Reuss.
 Introduction...
Electrophoresis is a separation method
based on the differential rate of migration of
charged species in a buffer solution across
which has been applied a dc electric field.
This separation technique was first
developed by the Swedish chemist Arne
Tiselius in the 1930s for the study of serum
proteins; he was awarded the 1948 Nobel
Prize for this work.
 On a macro scale, has been applied to a variety of
difficult analytical separation problems: inorganic
anions and cations, amino acids, etc.
A particular strength of electrophoresis is its
unique ability to separate charged macromolecules
of interest in biochemical, biological, and
biomedical research and the biotechnology
industry.
Performed by injecting a small band of the sample
into an aqueous buffer solution contained in a
narrow tube or on a flat porous support medium
such as paper or semisolid gel.
 Principles
There are several types of electrophoresis, but
the concepts are similar.
The machine has an anode (positive charge) and
a cathode (negative charge)
Negative ions move toward the anode and
positive charged ions move towards the cathode.
The rate and distance traveled by these
molecules help scientists classify and study
different biomolecules.
 Types ...

Electrophoretic separations are currently

performed in two quite different formats:
(1) slab electrophoresis
(2) capillary electrophoresis.
 Slab separations...

Slab separations are carried out on a thin

flat layer or slab of a porous semisolid gel
containing an aqueous buffer solution
within its pores. Ordinarily this slab has
dimensions of a few centimeters on a side
and, like a chromatographic thin layer plate,
is capable of separating several samples
simultaneously.
The Basis for Electrophoretic Separations...

The migration velocity v of an ion in

centimeters per second in an electric field
is equal to the product of the field
strength E (Vcm 1) and the
electrophoretic mobility ue(cm2V-1s-1).
That is
v = ue E
 Capillary Electrophoresis

Separates molecules based on their charge

and mass. Molecules are placed in rows
called capillaries filled with conductive
electrolyte fluid. The analytes move in a
speed relative to their charge and mass.
 Migration Rates and Plate Heights in
Capillary Electrophoresis

As we have already seen in the previous

equation, an ions migration velocity v
depends upon the electric field strength. The
electric field in turn is determined by the
magnitude of the applied potential (V, in
volts) and the length L over which it is
applied. Thus,
v = ue (V/L)
It has been shown for electrophoresis, that
the plate count (N) is given by
N = (ue*V/2D)

where D is the diffusion coefficient of the

solute in cm2s-1.
 Electro-Osmotic Flow...

When a high potential is applied across a

capillary tube containing a buffer solution,
Electroosmotic flow usually occurs, in
which the solvent migrates toward the
cathode or the anode. The rate of migration
can be appreciable.
The electroosmostic flow velocity v is given
by an similar equation
V = ueoE
In the presence of electroosmosis, an ions
velocity is the sum of its migration velocity
and the velocity of electroosmostic flow.
Thus,
V = (ue + ueo ) E
Note that ue for an anion will carry a negative sign.
Instrument for CE
 Instrumentation for CE
Sample Introduction
Electrokinetic injection
- one end of the capillary and its electrode are
removed from their buffer compartment and placed
in a small cup containing the sample.
Pressure injection
- the sample-introduction end of the capillary is
also placed in a small cup containing the sample,
but here a pressure difference drives the sample
solution into the capillary.
 Detection

Because the separated analytes move past a

common point in most types of CE,
detectors are similar in design and function
to those described for HPLC.
 Fluorescence Detection...
Just as in HPLC, fluorescence detection
yields increased sensitivity and selectivity
for fluorescent analytes or fluorescent
derivatives. Laser-based instrumentation is
preferred in order to focus the excitation
radiation on the small capillary and to
achieve the low detection limits available
from intense source. Laser fluorescence
detection has allowed detection of only 10
zeptomoles or 6000 molecules.
 Electrochemical Detection...

Two types of electrochemical detection

have been used with capillary
electrophoresis: conductivity and
amperometry. One of the problems with
electrochemical detection has been that of
isolation the detector electrodes from the
high potential required for the separation.
 Mass Spectrometric Detection...

The very small volumetric flow rates of

under 1 uL .min from electrophoresis
capillaries makes it feasible to couple the
effluent from the capillary of an
electrophoretic device directly to the
ionization source of a mass spectrometer.
 APPLICATION OF CAPILLARY
ELECTROPHORESIS

Capillary electrophoretic separations are

performed in several ways called modes.
These modes include:
capillary zone electrophoresis (CZE),
capillary gel electrophoresis (CGE),
capillary isoelectric focusing (CIEF), and
capillary isotachophoresis (CITP).
 CAPILLARY ELECTROCHROMATOGRAPHY

Electrochromatography is a hybrid of
capillary electrophoresis and HPLC that
offers some of the best features of the two
techniques. Two types of capillary
electrochromatography have been
developed since the early 1980s: packed
column and micellar electrokinetic capillary