Chapter 30

Capillary Electrophoresis and

Capillary Electrochromatography
 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.
• Electrophoresis has been applied to a
variety of difficult analytical separation
problems: inorganic anions and cations,
amino acids, catecholamines, drugs, nucleic
acids, nucleotides, polynucleotides, and
numerous other. A particular strength of
electrophoresis is its unique ability to
separate charged macromolecules of
interest in the biotechnology industry and in
biochemical and biological research.
 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
 Migration Rates and Plate Heights in
Capillary Electrophoresis

As we have already seen in the previous

equation, an ion’s 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 ion’s
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.
 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
 References...

• http://www.acs.org
• http://www.cas.org
• http://www.chemcenter/org
• http://www.sciencemag.org
• http://www.kerouac.pharm.uky.edu/asrg/
wave/wavehp.html