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现代仪器分析引论 Powered By Docstoc
					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
Slab electrophoresis
        Professor Stellan Hjertén
  The father of capillary electrophoresis

The first instrument of
capillary electrophoresis
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.


                                                 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;
•   Sample loading: pressure; voltage.

                                                          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

4. Separating the fragments by CE

5. Read the sequences
The automation of DNA sequencing, 96 channels.

How fast can we do it?

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