HPLC (IEX, Affinity and size)

Question 1

What mechanism does ion exchange chromatography separate components on

Answer 1

electrostatic attractions to the stationary phase

Question 2

motto that goes with ion exchange chromatography

Answer 2

opposites attract

Question 3

anion exchange chromatography binds what kind of proteins

Answer 3

anionic proteins

Question 4

describe the stationary phase of anion chromatography

Answer 4

positively charged

Question 5

describe the stationary phase of cation exchange

Answer 5

negatively charged

Question 6

how are the proteins extracted from the column

Answer 6

salt solution or change buffers

Question 7

how does adding a salt solution cause proteins to elute

Answer 7

the Cl- or NA+ displaces the protein due to the greater electronegativity

Question 8

how is the salt then removed from the protein

Answer 8

dialysis
-osmosis causes water to migrate out

Question 9

if a protein requires more salt to elute what can be said about the charges on the protein

Answer 9

more salt means a greater electrical charge

Question 10

how does changing buffer solutions cause protein elution

Answer 10

changing the pH of the solution can change the charges on the protein resulting in repulsion from the stationary phase

Question 11

anion exchange buffers

Answer 11

basic amines
-L-histidine
-bis-tris
-triethanolamine

Question 12

cation exchange buffers

Answer 12

acids
-formate
-acetate
-MES

Question 13

advantages to ion exchange chromatography

Answer 13

non-denaturing
use for almost any kind of charged molecule
useful for all stages of purification (incl concentration)
can be controlled by changing the pH, salt, concentration, and exchange media
high selectivity

Question 14

Disadvantages of ion exchange chromatography

Answer 14

costly

Question 15

principle of affinity chromatography

Answer 15

separation based on molecular features and highly specific interactions

Question 16

use and advantages to affinity chromatography

Answer 16

used for molecules with biological activity.

Has high selectivity and resolution.
Can separates and concentrate compounds in one step.

Question 17

steps in affinity chromatography

Answer 17

bind
wash
elute

Question 18

size exclusion chromatography principle

Answer 18

separation based on size and molecular weight move through the stationary phase at different rates

Question 19

types of stationary phase used in size exclusion

Answer 19

porous silica
porous agarose beads
monolith

Question 20

difference between gel:
filtrations
permeation

Answer 20

filtration: aqueous solution used as a mobile phase
permeation: organic solvent used as a mobile phase

Question 21

describe the buffer of size exclusion chromatography

Answer 21

it is run isocratically
(run the same throughout)

Question 22

how does size exclusoin chromatography work

Answer 22

small molecules diffuse into large and small pores resulting bin slower elution meanwhile large molecules flow through

Question 23

advantages to size exclusion

Answer 23

molecules don’t bind to the medium so buffer composition does not directly affect the resolution
suited for biomolecules that are pH, salt, and solvent-sensitive
can be used after HPLC as the components of buffer don’t affect final separation

Question 24

disadvanatges to size exclusion

Answer 24

results in the dilution of the separated sample
may need large columns
cannot distinguish between molecules of similar molecular mass (enantiomers)

Question 25

examples of molecules used for affinity chromatography

Answer 25

-Antigen and antibody
-Protein and nucleic acid
-ligand and receptor