# 现代仪器分析引论

```					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
(1902－1971）
Slab electrophoresis
Professor Stellan Hjertén
The father of capillary electrophoresis

The first instrument of
capillary electrophoresis
(1967)
The important town of modern separation techniques
Uppsala University
CE is running in a buffer filled,
narrow-bore capillaries;

The capillary is 25-100 μm in
internal diameter;
Light source
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
Electroosmotic flow

• Double electric layers and the ζpotential.
• In the case of fused-silica capillary, the electroosmotic
flow is always towards to anode.

potential

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
ν0 =νeof

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

plug                laminar

Mobile phase driven by electroosmotic flow in CE
and by pressure-induced flow in HPLC
CE instrumental
•   Power supply: 5～30 kV；
•   Capillary: fused-silica capillary, 50 µm and 75 µm i.d.
•   Detector: UV-Vis; Fluorescence; Mass spectrometry;

Fused-silica
Tube opening
Polymer film
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
polymerase

4. Separating the fragments by CE