Electrophoresis 3

Question 1

What is capillary electrophoresis?

Answer 1

electrophoresis that can be performed without a supporting media
- can be carried out in free buffer (only contains sample and buffer)

free buffer is useful as
- heat dissapates quickly so higher voltage can be used to see better separation

Question 2

How is separation carried out in capillary electrophoresis?

Answer 2

• a capillary joins the two buffers
• voltage moves through the capillary
• separation occurs within the capillary
• detector detects the compounds as they move through the capillary

separation is based on difference in analyte velocity in an electric field

Question 3

What are the advantages of using capillary electrophoresis?

Answer 3

high efficiency 
good reproducibility 
selectivity 
- can confirm drug purity and be used to chiral separation
works over a broad pH range 
fast analysis 
- can separate many compounds in a short time
can cope with a very small sample size

Question 4

What is the column made up of in capillary electrophoresis?

Answer 4

narrow bore fused silica column coated with polyimide

- polyimide is for protection = prevents the column from breaking

Question 5

What is electroosmotic flow?

Answer 5

buffer flows towards the cathode (negative electrode)

this happens because

• the column is made up of glass and silica. the silica has silanol groups (SiOH) which ionise at most pHs
• if an electric field is applied, the positive cations (H+) move towards the cathode whereas the negative anions (SiO-) are stuck to the column
• results in a greater concentration cations than anions leading to bulk flow towards the cathode

Question 6

Why does electroosmotic result in one way flow? Why is this advantageous?

Answer 6

the greater concentration of cations than anions means that they cause the flow of ions to be in their direction of movement
- towards the cathode
= forms an electrical double layer with the negatively charged columns

means that only one detector is needed at one end of the capillary

Question 7

How is capillary electrophoresis used for chiral separations?

Answer 7

can be difficult to separate due to their identical physical and chemical properties

must provide a chiral environment by adding a chiral selector to the buffer (cyclodextrin)
- enantiomer placed in will form temporary diastereoisomers which can be separated as they have different mobilities

Question 8

What are the factors determine resolution of chiral separations?

Answer 8

chiral selector concentration
buffer concentration
temperature