Chapter 13 Capillary electrophoresis
Electrophoresis: an overview
Definition: a separation method based on the differential
movement for migration of charged species by attraction or
repulsion in an electric field.
= eE = eV/L
velocity of molecule
E electric field
e electrophoretic mobility
V applied voltage
L the distance between electrodes
Can determine the size, shape, and charge of a molecule.
Types of electrophoresis
• Capillary electrophoresis (CE; also HPCE)
• Native polyacrylamide gel electrophoresis (native PAGE)
• SDS polyacrylamide gel electrophoresis (SDS-PAGE)
• Slab electrophoresis
• Paper electrophoresis
• Free electrophoresis
Electrophoresis: the history
In1940, Tiselius used the device below
and successfully separated human serum
proteins into 4 components. He named them
as albumin, α,β,γglobulin.
Arne W.K. Tiselius
Professor Stellan Hjertén
The father of capillary electrophoresis
The first instrument of
The important town of modern separation techniques
CE is running in a buffer filled,
The capillary is 25-100 μm in
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 so that separation is achieved.
The factors affecting on e
Schematic of a CE system
• Double electric layers and the ζpotential.
• In the case of fused-silica capillary, the electroosmotic
flow is always towards to anode.
Distance from the wall
Total mobility of ions in a fused-silica capillary
The rate of electroosmotic flow is much greater than the
electrophoretic migration velocities of all ions in solution.
The total mobility of ions in CE:
Question: how to measure the
ν+ =νeof + νef flow rate of electroosmosis?
ν- =νeof - νef
All ions are then swept toward the anode.
Negative ions will lead the neutral species toward the anode.
Positive ions will trail the neutral species as the cathode pulls them.
The flow profile of electroosmotic flow
Mobile phase driven by electroosmotic flow in CE
and by pressure-induced flow in HPLC
• Power supply: 5～30 kV；
• Capillary: fused-silica capillary, 50 µm and 75 µm i.d.
• Detector: UV-Vis; Fluorescence; Mass spectrometry;
• Sample loading: pressure; voltage.
Applications of CE: basic operating modes
• Capillary zone electrophoresis (CZE)
• Capillary gel electrophoresis (CGE)
• Micellar electrokinetic capillary chromatography (MECC）
• Capillary isoelectric focusing (CIEF)
• Capillary isotachophoresis (CITP)
Applications of CE: DNA sequencing
Two key techniques were relied on in nucleic acid sequencing
1. Restriction endonuclease
2. Polyacrylamide gel electrophoresis
The basic protocol
1. dideoxy method for chain termination
2. base-specific cleavage of the chain
The steps for DNA sequencing
1. Templet ssDNA from dsDNA
2. Where is the starting point? The
use of primer
3. Synthesize new ssDNAs on the
templet ssDNA, elongate from
5′→3′, by the aid of DNA
4. Separating the fragments by CE
5. Read the sequences
The automation of DNA sequencing, 96 channels.
How fast can we do it?