1

Basic principles
Movement of sample ions under the influence of an applied
voltage.
=

C
s2 A
=
=

6
Pa s m
kg

s2 A kg m2
m
= =
3
=
kg s A m
s

Electric
charge carrier
mobility

Ion migration
rate

Basic principles
Forces acting on a charge moving in electrical field.

= + + = 0

Steady-state movement

https://lcp.elis.ugent.be/research/electrophoresis

Basic principles
Electrophoresis in free flowing solution (moving boundary
electrophoresis) inefficient separation
Suitable for huge molecules
Molecules with poor diffusion

Therefore zone electrophoresis is used (electrophoresis using

supporting medium):

Electrophoresis in paper matrix

Electrophoresis in cellulose acetate strips
Electrophoresis in gel matrix
Electrophoresis in thin capillary

Basic principles
Another driving force zone electrophporesis is the
electroosmotic flow.
For example, if silica capillary is used, at pH where large part
of silanol groups are ionized, ions in the diffuse layer of the
formed electrical double layer will migrate towards cathode
and drag solvent along.

http://en.wikipedia.org/wiki/File:Electroosmotic_Flow_Depiction.tiff

Basic principles
Electroosmotic flow depends on zeta potential

http://upload.wikimedia.org/wikipedia/commons/thumb/0/05/Diagram_of_zeta_potential_and_slipping_planeV2.svg/

Basic principles
Electroosmotic flow causes a flat flow profile
(also plug flow)

http://microsolvtech.com/images/cebasic1.gif

Process description
Migration time

Peak efficiency by the number of plates

2

=
=

2
Resolution
=

= 0.177 2 1

1,2
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Apparatus for electrophoresis on

supporting medium
Stabilized high voltage DC current source
Indicator and voltage adjustment control
Electrodes
Electrophoresis chamber
Supporting medium

Conducting liquid or buffer

Thermostating (buffer circulation, air colling, etc.)
Detector or staining
9

Gel electrophoresis
Useful for separation and studies of oligonucleotides, DNA, RNA, proteins.
Separation by:
pI (isoelectric point)
Mass-to-charge
Thermal properties

Under different
conditions:

Denaturing
Native

Separation in:
Agarose gel
Polyacrylamide gel
Starch gel

Under native conditions proteins exist in their folded state and the
separation depends both on charge-to-mass ratio and shape-size factors.
Isoelectric focusing is size independent.
Transfer of analytes after separation is possible.

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Gel electrophoresis

http://openwetware.org/images/thumb/7/7b/Be109agarosegelEM.jpg/250px-Be109agarosegelEM.jpg

11

Gel electrophoresis
Useful for separation and studies of oligonucleotides, DNA,
RNA, proteins.

http://upload.wikimedia.org/wikipedia/commons/4/46/SDS-PAGE_Electrophoresis.png
12

Gel electrophoresis

http://www.tankonyvtar.hu/en/tartalom/tamop425/0011_1A_Proteinbiotech_en_book/ch05.html
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2D gel electrophoresis
The separation of analytes
first by one property and
then in the perpendicular
direction by another
property.
Often first separated by
isoelectric point,
afterwards treated with
denaturing agent (to
eliminate shape) and
separated by molecular
mass.

Spot picking is possible

for further analysis, e.g.,
MALDI-TOF.

http://www.tjbiochip.com/upload/fckeditor/-.jpg

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Capillary electrophoresis

Maria Luisa Marina, Angel Ros, Miguel Valcrcel Analysis and

Detection by Capillary Electrophoresis
15

Capillary electrophoresis
CE HPLC

http://microsolvtech.com/cebasic.asp
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Capillary electrophoresis buffers

High buffer capacity in pH range chosen.

Low response towards the detector.
Low mobility to prevent current generation.
Available in high purity.
Low temperature coefficient for pH/deg.

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Capillary electrophoresis buffers

Maria Luisa Marina, Angel Ros, Miguel Valcrcel Analysis and Detection by Capillary Electrophoresis
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Electroosmotic flow modifiers

Maria Luisa Marina, Angel Ros, Miguel Valcrcel Analysis and Detection by Capillary Electrophoresis
19

Capillary zone electroforesis

Most widespread
technique.
Capillary contains
only buffer.
Separation
according to massto-charge ratio.

Maria Luisa Marina, Angel Ros, Miguel Valcrcel Analysis and Detection by Capillary Electrophoresis
20

Influencing parameters

Maria Luisa Marina, Angel Ros, Miguel Valcrcel Analysis and Detection by Capillary Electrophoresis
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THE END

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