Gel and Ion exchange Chromatography methods

purpose of gel filtration chromatography and method

purify proteins based on size

seperates complex protein mix as different size proteins migrate at different speeds

prerequisite for gel filtration

must have approximae idea od protein size

helps predict elution volume/time on column

how size seperation works

pore structure of the SP is altered

pore size is carefully controlled (mw cutoff)

allows proteins below a certain size to enter the beads larger proteins excluded

void volume

volume outside the beads where large proeins unable to enter pores travel faster

internal volume

volume wihtin the beads (inside pores) that small proteins can enter

exclusion limit

mw cutoff above whihc proteins are too large to enter pores of SP

permeation link

cutoff below which proteins are small enough to enter all pores

migration of proteins above exclusion limit

only travel thorugh void volume so they elute faster

migration of proteins below permeation limit

much longer path

elute slower

intermediate region

size range in between limits

proteins can access some but not all internal volume

intermediate region produces what relationship?

log linear relationship between MW and elution volume allowing for good seperation and resolution

ion exchange chromatography exploits what

charge differences of proteins at given pH

type types of ion exchange chromatography and their modifications

beads are not particulary porous

appended with charged molecular species

anion exchange = positve charges binds (-) proteins

cation exchange = negative charges binds (+) proteins

mobile phase in ion exchnage

contains salt (e.g. chloride ions) that compete with proteins for binding sites

ion exchange process

1. protein is injected

2. proteins with net charge opposite to that of SP bind to the column

3. elution

   1. salt gradient applied by gradually increasing the [salt] in the mobile phase

how does [salt] affect elution

weakly bound proteins elute at low [salt]

strongly bound proteins require higher [salt] to be displaced

elution peaks for ion exchange

early eluting peaks (left) represent proteins that don’t bind strongly

later peaks correspond to proteins that are bound more tightly —> require higher [salt] for elution

ion exchange for analyical approach

can run an already pure protein to determine properties like monomer vs dimer

net charge of proteins depends on their __

pH relative to their pl

describe the salt gradient effect on proteins in ion exchange

as [salt] increases it weaken electrostatic interactions between proteins and SP

causes proteins to elute in order of increasing binding strength i.e. more (-) proteins require higher [salt] to elute in an ANION exchange setup