1

Basic principles

Movement of sample ions under the influence of an applied voltage.

=

=

6=

C

Pa s m

s2 A

kg

= =s2 A

kgkg m2

s3 A m=

m

s

Electric

charge carrier

mobility

Ion migration

rate

2

Basic principles

Forces acting on a charge moving in electrical field.

= + + = 0 Steady-state movement

https://lcp.elis.ugent.be/research/electrophoresis 3

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

4

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.

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

Basic principles

Electroosmotic flow depends on zeta potential

=

4

6 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)

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

Process description

Migration time

=

Peak efficiency by the number of plates

=

2

=

2

Resolution

=

4

= 0.177 2 1

1,2

8

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.

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.

10

Separation by:

pI (isoelectric point)

Mass-to-charge

Thermal properties

Under different conditions:

Denaturing

Native

Separation in:

Agarose gel

Polyacrylamide gel

Starch gel

Gel electrophoresis

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

Gel electrophoresis

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

12

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

Gel electrophoresis

13

http://www.tankonyvtar.hu/en/tartalom/tamop425/0011_1A_Proteinbiotech_en_book/ch05.html

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.

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

Capillary electrophoresis

15

Maria Luisa Marina, Angel Ros, Miguel Valcrcel Analysis and

Detection by Capillary Electrophoresis

Capillary electrophoresis

16

http://microsolvtech.com/cebasic.asp

CE HPLC

Capillary electrophoresis buffers

17

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.

Capillary electrophoresis buffers

18

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

Electroosmotic flow modifiers

19

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

Capillary zone electroforesis

20

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

Most widespread technique.

Capillary contains only buffer.

Separation according to mass-

to-charge ratio.

Influencing parameters

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

THE END

22