HPLC (IEX, Affinity and size) Flashcards
What mechanism does ion exchange chromatography separate components on
electrostatic attractions to the stationary phase
motto that goes with ion exchange chromatography
opposites attract
anion exchange chromatography binds what kind of proteins
anionic proteins
describe the stationary phase of anion chromatography
positively charged
describe the stationary phase of cation exchange
negatively charged
how are the proteins extracted from the column
salt solution or change buffers
how does adding a salt solution cause proteins to elute
the Cl- or NA+ displaces the protein due to the greater electronegativity
how is the salt then removed from the protein
dialysis
-osmosis causes water to migrate out
if a protein requires more salt to elute what can be said about the charges on the protein
more salt means a greater electrical charge
how does changing buffer solutions cause protein elution
changing the pH of the solution can change the charges on the protein resulting in repulsion from the stationary phase
anion exchange buffers
basic amines
-L-histidine
-bis-tris
-triethanolamine
cation exchange buffers
acids
-formate
-acetate
-MES
advantages to ion exchange chromatography
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
Disadvantages of ion exchange chromatography
costly
principle of affinity chromatography
separation based on molecular features and highly specific interactions
use and advantages to affinity chromatography
used for molecules with biological activity.
Has high selectivity and resolution.
Can separates and concentrate compounds in one step.
steps in affinity chromatography
bind
wash
elute
size exclusion chromatography principle
separation based on size and molecular weight move through the stationary phase at different rates
types of stationary phase used in size exclusion
porous silica
porous agarose beads
monolith
difference between gel:
filtrations
permeation
filtration: aqueous solution used as a mobile phase
permeation: organic solvent used as a mobile phase
describe the buffer of size exclusion chromatography
it is run isocratically
(run the same throughout)
how does size exclusoin chromatography work
small molecules diffuse into large and small pores resulting bin slower elution meanwhile large molecules flow through
advantages to size exclusion
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
disadvanatges to size exclusion
results in the dilution of the separated sample
may need large columns
cannot distinguish between molecules of similar molecular mass (enantiomers)
examples of molecules used for affinity chromatography
-Antigen and antibody
-Protein and nucleic acid
-ligand and receptor
