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Question 1

Types of chromatographic methods

Answer 1

1) Size-exclusion chromatography (Gel Chromatography)
2) Ion-exchange chromatography
3) Reversed phase chromatography
4) Hydrophobic interaction chromatography
5) Affinity chromatography

Question 2

Main stages of chromatography columns

Answer 2

1) Equilibration (column preparation)
2) Sample application and wash (wash away unbound proteins/contaminants)
3) Elution (elute out desired bound proteins)
4) Regeneration (regenerate column for next cycle)

Question 3

Principle of size-exclusion chromatography

Answer 3

separation based on molecular size

1) Large molecules are completely excluded from gel matrix and move rapidly through the column and are eluted first.
2) Small molecules penetrate the pores of packed column and transverse slowly and are eluted last

Question 4

Factors that affect SEC

Answer 4

1) Matrix
- pore size
- pore volume
- rigidity
- chemical stability
- bead size
- bead size distribution
2) Bed height
3) Column packing
4) Flow velocity
5) Sample
6) Volume

Question 5

Principles of Ion-exchange chromatography

Answer 5

• utilises polarity protein
• based on relative affinities of components for ionic sites
• elution occurs by changing the pH or ionic strength

Question 6

Ion-exchange media

Answer 6

• made from porous or non-porous matrices covered by charged groups
• common ion exchange matrices:
  1) polystyrene with divinyl benzene as cross-linker
  2) agarose
  3) cellulose

Question 7

Base matrix for ion exchange

Answer 7

1) hydrophilic
2) large pore size
3) spherical particle
4) mono-size
5) rigid
6) chemically stable

Question 8

Types of IEC

Answer 8

1) Cation exchanger (negatively charged resin)
2) Anion exchanger (positively charged resin)

Question 9

How to choose an IEC resin?

Answer 9

• Binding is dependent on pH
• at ph below the isoelectric point of a target, net charge is positive
• binds to a cation exchange resin
• at pH above the isoelectric point, net charge is negative
• binds to anion exchange resin

Question 10

Sample elution Mechanism of IEC

Answer 10

• sample eluted from the resin by displacement with another ion that has a stronger affinity for solid matrix
• Cl^- for anion exchanger and Na^+ for cation exchanger

Question 11

Mechanism of IEC

Answer 11

Solid matrix contains covalently attached positive groups (anion exchanger) or negatively charged groups (cation exchanger)

Question 12

Sample application and wash

Answer 12

• charged regions of the protein containing a charge of opposite polarity to the resin will be attracted to the solid matrix
• the counter ion originally attached to the resin will be replaced by the charged protein
• washing separates out unbound non-charged species

Question 13

Principles of Hydrophobic interaction chromatography

Answer 13

• utilises protein hydrophobicity
• separation based on attraction between hydrophobic groups on protein and hydrophobic matrix
• HIC is less hydrophobic than RPC
• Sample applied under high salt concentration and eluted under low salt concentration
• the more hydrophobic the protein, the tighter the binding

Question 14

How does separation occur in HIC?

Answer 14

• Weak hydrophobic interaction between protein and matrix
• Hence, sample is applied under high salt concentration and eluted under low salt concentration
• Addition of salt increases the ionic strength of solution, hence increasing hydrophobicity of protein molecules.
• This repels water molecules away from protein molecules, helping unmask hydrophobic domains on protein surface.

Question 15

Base matrix of HIC

Answer 15

• consists of a hydrophobic polymer (styrene cross-linked with divinylbenzene)
• OR a hydrophilic polymer (agarose, dextran etc.) with a hydrophobic group attached
• Common groups in order of increasing hydrophobicity:
  Butyl < Octyl < Phenyl

Question 16

Mechanism of HIC

Answer 16

• solid matrix contains weak hydrophobic groups
• column equilibration with buffer to prepare column for use

Question 17

Mechanism of HIC for sample application&wash

Answer 17

• sample is applied in high salt concentration
• addition of salt increases ionic strength of solution thus increasing the hydrophobicity of protein molecules
• water molecules repelled away from protein molecules thus unmasking hydrophobic domains on protei surface
• hydrophobic proteins bind reversibly to hydrophobic matrix
• washing separates out unbound contaminants

Question 18

Mechanism of HIC for sample elution

Answer 18

• sample eluted in low salt concentration
• decrease in salt concentration decreases hydrophobicity of proteins, causing proteins to elute from the column

Question 19

Column regeneration of HIC

Answer 19

Regeneration of the resin with buffer solution to wash away all residual contaminants in preparation of next cycle

Question 20

Advantages of HIC

Answer 20

1) Concentrates sample
2) Relatively low cost
3) High capacity
4) High speed
5) High resolution

Question 21

Limitations of HIC

Answer 21

• high salt concentrations may generate high back pressure on column

Question 22

Applications of HIC

Answer 22

• hydrophobic proteins (8 of 20 amino acids are hydrophobic)
• most suitable when ionic strength of sample is high (eg. after affinity chromatography)

Question 23

Principles of Affinity chromatography

Answer 23

• utilises bio recognition/ ligand specificity
• separation is based on specific and reversible binding of biomolecules to the matrix
• conditions for elution depend on the the specific binding complex formed
• elution usually involves a change in pH, ionic strength or buffer composition

Question 24

Base matrix of affinity chromatography

Answer 24

column packing is prepared by linking a binding molecule called a ligand to an insoluble support

Question 25

Chelating agents

Answer 25

-chemical compounds that react with metal ions to form a stable complex