ELECTROPHORESIS
= an analytical method based on a movement of charged particles because of an external electric field
electrophoretic mobility
- mobility of a substance (i.e. how quickly an ion moves in an electric field)
- it depends on the:
a) size, shape and charge of the substance
b) given applied voltage
anion - negatively charged ion, it moves to the anode (+)
cation - positively charged ion, it moves to the catode (-)
amphoteric - a substance that can have a positive, zero, or negative charge, depending on conditions
(e.g. proteins)
classification of electrophoresis
1) free-boundary e. - separation is carried out entirely in a liquid phase, i.e. no support is used
(capillary electrophoresis)
2) e. in a supporting medium - paper, gel (agarose, polyacrylamide)
- it can be done horizontally or vertically
effects of electrophoretic parameters on separation
pH
- changes charge of analyte and hence its mobility
- can affect structure of analyte (denaturing, dissociating)
ionic strength - changes voltage or current: increased ionic strength usually reduces migration velocity
and increases heating
temperature - overheating can denaturate (precipitate) proteins
- lower t. reduce diffusion but also reduce migration velocity, no effect on resolution
current
- too high current causes overheating
voltage
- migration velocity is proportional to voltage
time
- resolution (separation of bands) increases linearly with time, but dilution of bands (diffusion)
increases with the square root of time
medium
- major factors are endosmosis and pore-size effects, which affect migration velocities
process of electrophoresis
1) sample application
2) adjustment of voltage or current (!!direct current !!)
(gel-electrophoresis about 70 volts, capillary electrophoresis about 20,000 volts)
3) separation time: minutes (e.g. gel-electrophoresis of serum proteins 30 min.)
4) electrophoresis in supporting medium: fixation, staining
5) evaluation: * qualitative (standards)
* quantitative (densitometry)
application examples:
* separation of serum proteins, isoenzymes, nucleic acids
* imunoelectrophoresis (immunoglobulins)