Electrophoresis

Question 1

What is electrophoresis

Answer 1

Incomplete type of electrolysis (stops before molecules reach electrodes)

Biological molecules possess ionisable groups; in solution possess an electric charge:
- Amino acids, peptides & proteins.
- Nucleotides & nucleic acids.

Nature of electric charge is pH-dependent.

Positively-charged molecules: Cations. Migrate to negative cathode.

Negatively-charged molecules: Anions. Migrate to positive anode.

Question 2

What equipment is used in electrophoresis

Answer 2

Power pack: provides electrical power → Movement of charged molecules

Electrophoresis unit:
- Vertical gel systems. Separates proteins in acrylamide gels
- horizontal gel systems. Separate DNA in agarose gels.

Buffer solution: Specific pH maintains electrical charge & analyte molecule mobility.

Question 3

What is potential difference and what does it do

Answer 3

Difference in electrical potential energy between the electrodes. Measured in Volts (V).

Potential Difference allows a current of charged molecules to flow:
cathode → anode AND anode → cathode (simultaneously both ways)

Question 4

What is the Frictional Coefficient.

Answer 4

The Frictional resistance acting against the force of movement

Depends on:
Hydrodynamic size & shape of molecule.
Pore size of electrophoresis medium (the gel).
Buffer viscosity.

Question 5

How do you calculate the velocity of a molecule

Answer 5

Potential Difference × Charge on Molecule
————————————————————————
Distance between Electrodes × Frictional Coefficient

Question 6

What affects the velocity of a molecule

Answer 6

electrical charge
molecular size (more/less friction)

Question 7

How is separation completed in electrophoresis

Answer 7

Molecules with different velocities located at different distances from electrodes = SEPARATION!

Smallest DNA fragments have greatest velocity;
largest DNA fragments have least velocity.