Chromatography

Question 1

What is chromatography?

Answer 1

Means of separating and identifying compounds and mixtures.
Broad range of physical methods used to separate and or analyse complex mixtures

Question 2

Phases of chromatography

Answer 2

Components to be separated fare distributed between 2 phases -
1. Stationary phase bed
2. Mobile phase which percolates through the stationary bed.

Question 3

Principles of chromatography

Answer 3

Separation of molecules based on structural or composition differences

Molecules have different interactions with the stationary support which leads to separation

Molecules which display tighter interactions with the support move slower

Question 4

What are the different types of liquid chromatography?

Answer 4

Size exclusion
Ion exchange
Hydrophobic interaction
Normal phase
Reverse phase
Affinity

Question 5

Size exclusion chromatography basis of separation

Answer 5

Molecular size - larger molecules elude first

Question 6

Ion exchange chromatography basis of separation

Answer 6

Charge differences - anions/cations

Question 7

Hydrophobic interaction chromatography basis of separation

Answer 7

Hydrophobicity - more hydrophobic elude later

Question 8

Normal phase liquid chromatography basis of separation

Answer 8

Polarity - polar compounds elude last

Question 9

Reverse phase liquid chromatography basis of separation

Answer 9

Polarity - non polar compounds elute last

Question 10

Affinity chromatography basis of separation

Answer 10

Specific binding between target molecule and ligand

Question 11

Stationary phase of size exclusion

Answer 11

Porous beads - silica or polymers

Question 12

Stationary phase of ion exchange

Answer 12

Charged groups attached to resin

Question 13

Stationary phase of hydrophobic interaction

Answer 13

Hydrophobic ligands on the resin

Question 14

Stationary phase of normal phase

Answer 14

Polar eg silica

Question 15

Stationary phase of reverse phase

Answer 15

Non polar eg C18

Question 16

Stationary phase of affinity

Answer 16

Ligands eg antibodies, enzymes bound to matrix

Question 17

Mobile phase of size exclusion.

Answer 17

Aqueous or organic solvents

Question 18

Mobile phase of ion exchange.

Answer 18

Buffered aqueous solutions with varying ionic strength or pH

Question 19

Mobile phase of hydrophobic

Answer 19

Aqueous mobile phase with decreasing salt concentratations

Question 20

Mobile phase of normal phase

Answer 20

Non polar solvents eg hexane

Question 21

Mobile phase of reverse phase

Answer 21

Polar solvents eg water with methanol

Question 22

Mobile phase of affinity

Answer 22

Buffer solutions

Question 23

Sample interaction in size exclusion.

Answer 23

No direct interaction - separated by size

Question 24

Sample interaction in ion exchange.

Answer 24

Electrostatic interactions between charged sample and stationary phase.

Question 25

Sample interaction in hydrophobic

Answer 25

Hydrophobic interactions between sample and stationary phase

Question 26

Sample interaction in normal phase.

Answer 26

Polar compounds interact more with stationary phase.

Question 27

Sample interaction in reverse phase

Answer 27

Non polar compounds interact more with stationary phase

Question 28

Sample interaction in affinity

Answer 28

Specific molecular recognition eg antigen antibody

Question 29

Applications of size exclusion.

Answer 29

Protein purification, polymer analysis

Question 30

Applications of ion exchange.

Answer 30

Separation of proteins, nucleotides, charged molecules

Question 31

Applications of hydrophobic

Answer 31

Protein purification based on surface hydrophobicity

Question 32

Applications of normal phase.

Answer 32

Separation of polar compounds and small molecules

Question 33

Applications of reverse phase

Answer 33

Drug analysis Peptide separation Non polar organic molecules

Question 34

Applications of affinity

Answer 34

Purification of proteins, antibodies and enzymes