Electrophoresis Flashcards
What happens when an electric field is applied?
Charged species begin to move
Where do cations move towards?
Cathode
What does the rate of migration depend on? (x3)
- Net charge of molecule
- Size and shape of molecule
- External environment (buffer pH, electric field and temperature)
What is electrophoretic mobility defined as?
The average velocity with which an ion moves in an applied electric field (=V/E)
What does the electric force (Fe) depend on?
The charge and the (applied voltage/distance between electrodes)
What is movement opposed by, and what is this proportional to?
Frictional drag - exerted on the charged particles by the medium and is proportional to the velocity of the particle
What happens once electrophoresis has started?
There is rapid acceleration of the ions and equilibrium is reached within milliseconds
What happens to the resultant force at equilibrium?
It is 0 and the molecules move at a constant velocity
What can mobility be interpreted as?
Proportional to charge-to-size ratio
Describe the setup in capillary electrophoresis
A fused-silica capillary with an optical viewing window, a high voltage power supply, two electrodes, two buffer reservoirs and a detector
What molecules in particular can be separated in CE?
Polar molecules
What is the capillary filled with?
Buffer
How is the sample injected in CE?
Electrokinesis or pressure
What happens after electric field is applied in CE?
An electric double layer is produced at the capillary surface due to the attraction of positively charged ions in the buffer to the ionised silanol groups at the capillary wall
In the presence of an electric field, what happens to the cations in the diffuse (outer) portion
Move to the cathode and drag the solvent with them, producing an electroosmotic flow (EOF)
What is formed due to different electrophoretic mobilities of ionic species?
Zones of analytes that migrate toward the outer side of the capillary at different rates
What does rate of migration depend on in CE?
Mobility is proportional to q/f, which is approximately equal to the m/z ratio
What does electrophoresis refer to in GE?
The electromotive force (EMF) that is used to move molecules through a gel matrix
What is the gel?
A matrix used to separate proteins
What is the gel composed of?
Different concentrations of acrylamide and a cross-linker, forming a solid, porous matrix
What is the first step in GE?
Prepare stacking gel (large pores, made with Tris buffer 2 pH units below that of electrophoresis buffer) and resolving/separating gel (smaller pores, pH ~8.8, proteins separated by size)
What does the buffer contain in GE?
Glycerol, bromophenol and SDS + molecular weight standard proteins
What follows electrophoresis in GE?
Stain polyacrylamide gel to view protein bands
What is a gradient gel?
Rather than uniform pore size throughout the gel a gradient is formed by varying acrylamide concentrations
