lecture 3

Question 1

What are the 3 main stages to perform ion exchange chromatography?

Answer 1

• Equilibrium
• Sample application and wash
• Elution

Question 2

What does the matrix contain for ion exchange chromatography?

Answer 2

matrix contains ionised groups

Question 3

What happens when a proteins pI is above a pH?

Answer 3

It has an overall negative charge and will bind to a matrix that is positively charged

Question 4

What is an anion exchanger?

Answer 4

itself is positive

- Binds anions

Question 5

What is a cation exchanger?

Answer 5

• itself is negative

- Binds cations

Question 6

what is the strength of the exchanger proportional to?

Answer 6

• proportional to the net charge

Question 7

What is the matrix made of in ion exchange chromatography?

Answer 7

organic molecules

Question 8

What is a weak anion exchanger?

Answer 8

Diethylamino ethyl (DEAE)

Question 9

What is a strong anion exchanger?

Answer 9

Quaternary Ammonium (Q)

Question 10

What is a weak cation exchanger ?

Answer 10

Carboxymethyl (CM)

Question 11

What is a strong cation exchanger?

Answer 11

Methyl sulphonate (S)

Question 12

What are the two key conditions needed for ion exchange chromatography?

Answer 12

• Low ionic strength buffer – bind

- pH to generate opposite charge to ion exchange resin – know your pI

Question 13

What is chromatofocusing?

Answer 13

elution by changing pH

Question 14

How does Chromatofocusing work?

Answer 14

• proteins with different pI separate as they pass through . molecules with the same isoelectric point are focused in narrow bands during the separation

Question 15

How can elution by changing the salt occur?

Answer 15

• change the salt concentrations
• The salt ions compete with the protein for the charges on the stationary phase
• don’t change the pH

Question 16

What types of molecules can elution by pH separate?

Answer 16

separate the protein isoforms

Question 17

What are the advantages of ion exchange chromatography?

Answer 17

• Load large volume, elute small

- High capacity, good resolution

Question 18

What are the disadvantages of ion exchange chromatography?

Answer 18

• Denaturation at pH extremes
• Product in high salt (tend to ppt)
• pH gradient technically difficult

Question 19

How does hydrophobic interaction chromatography work?

Answer 19

separation via differing surface hydrophobicity , this is promoted by ‘salting out’
- salting out exposes the hydrophobic parts of the protein

Question 20

How are proteins eluted in regards to hydrophobic interactions?

Answer 20

• elute in order of hydrophobicity

by interfering with the interaction ( decreasing salt, changing salt, pH , temp or detergent )

Question 21

What is the drawback of elution by changing the salt concentration?

Answer 21

• may salt out on the column and not bind to the column at all

Question 22

What are the parameters that need to be considered when using hydrophobicity?

Answer 22

• The ligand (: C4, C8 linear aliphatic chain, )

- Initial salt concentration

Question 23

What type of technique is chromatofocusing?

Answer 23

Ion exchange chromatography