Professor Chadwick: Electrophoresis

Question 1

What is the principle of electrophoresis?

Answer 1

• An electrical field can be used to separate out ions.
• The separation depends on the mobility of the ions.
• The mobility is directly proportional to the charge of the ions
• The mobility is inversely proportional to the size. Larger molecules move more slowly.

Question 2

What are the advantages of using a capillary during electrophoresis?

Answer 2

• To efficiently separate the solution, very large voltages are needed which would heat the solution.
• Using a thin capillary stops the convection currents and the large surface area increases heat dissipation

Question 3

What is Electrophoresis?

Answer 3

• Electrophoresis is the migration of charged colloidal particles/molecules under the influence of an applied electrical field.
• It is a method of separating substances (especially proteins) and analysing molecular structure on the rate of movement of each component in a colloidal suspension.

Question 4

What is electroosmosis?

Answer 4

• Electro-osmosis is the motion of polar liquid through a membrane or other porous structure under the influence of an applied electrical field.

Question 5

What is the streaming potential?

Answer 5

• The potential produced when a liquid is forced to flow through a capillary or porous solid.
• The movement of liquid over the surface of the solid produces an electric current, because the flow of liquid causes a displacement of mobile counter-ions with respect to the fixed charges on the solid surface..
• The applied potential necessary to reduce the net flow of electricity to zero is streaming potental.

Question 6

What is the sedimentation potential?

Answer 6

• Sedimentation potential occurs due to dispersed particles motion relative to the fluid under influence of gravity/centrifugation.
• This motion disrupts equilibrium symmetry of the particles double layer. Enveloping viscous flow around the particles drags ions of diffuse layer away from the particles.
• This causes slight displacement between surface charge and electric charge of the diffuse layer.
• The particle gains a dipole moment which generate the sedimentation potential.

Question 7

What is the electrical double layer?

Answer 7

• The electrical double layer is the structure that describes the variation of electric potential near a surface, and has a large bearing on the behaviour of colloids and other surfaces in contact with solutions.

Question 8

What is the Helmholtz model for the electrical double layer?

Answer 8

• The Helmholtz model forms an idea that there is a layer of positive ions on the negative electrode surface.
• a = separation of layer
• epsilon₀ = permittivity of free space
• epsilon = dielectric constant of solution.
• sigma = charge density of layer

Question 9

What is the Guoy-Chapman model for the electrical double-layer

Answer 9

• Now a is the closest approach. Forms the idea that there is a Boltzmann distribution of ion in the diffuse layer.
• Theory is similar to Debye-Huckel theory, thus d is the average thickness of the diffuse layer.
• The electric potential at the closest approach is known as the zeta potential.

Question 10

What is the Stern Model for the Electrical Double-Layer?

Answer 10

• Models a fixed double-layer and a diffuse double layer.
• This gives a better model for experiments.
• The zeta potential is the potential at the edge of the fixed region.

Question 11

What is the Zeta Potential?

Answer 11

• The zeta potential is the effective potential at the surface of the double layer.
• It is this potential that reacts with a voltage to produce a force: Force = charge density x voltage.

Question 12

What is capillary zone electrophoresis?

Answer 12

• CZE is most commonly used type of capillary due to simplicity and versatility.
• Applications are very diverse and can be used for peptide, ion and enantiomer analysis.
• Easiest form of CE as it is only filled with buffer.
• Separation occurs as solutes move at different velocities.
• Separates anions and cations in the same run due to electroosmosis which helps/hinders different analytes
• CZE cannot separate neutral molecules.

Question 13

What is capillary gel electrophoresis (CGE)?

Answer 13

• Adapts traditional gel electrophoresis into the capillary using polymers in solution to create a molecular sieve.
• This allows analytes having similar m/z ratios to be resolved by size.
• Commonly employed in SDS-Gel molecular weight analysis of proteins and sizing of applications of DNA sequencing and genotyping.

Question 14

What is the process of gel electrophoresis?

Answer 14

1. Restriction enzymes cleave the DNA into smaller segments of various sizes.
2. DNA segments are loaded into wells in a porous gel. The gel floats in a buffer solution within a chamber between two electrodes.
3. When an electric current passes through the chamber, DNA fragments move towards the positively charged anode.
4. Smaller DNA segments move faster and further than larger DNA segments.

Question 15

Comparison of CZE with Slab Gel Electrophoresis

Answer 15

1. SGE = cheaper equipment than CZE
2. CZE more sensitive than SGE
3. CZE has better resolution than SGE
4. CZE has a more simple and more precise sample introduction than SGE.
5. CZE has a better and faster readout than SGE as it is direct from detector to computor.
6. CZE uses higher voltages than SGE; quicker and less broadening of sample components.