Chapter 5 Ion Exchange Chromatography Flashcards
Objectives
Know the importance of ion exchange chromatography in protein purification.
Describe the process of ion exchange chromatography.
Understand how the pI and pH stability of a target protein determines its binding and elution strategies.
Know the elution strategies used in ion exchange chromatography.
Where is the chromatography step in the general workflow
Between protein mixtures and peptides
Ion exchange chromatography
- Initial stage
- absorption of target
- starting of elution
- end of elution
- regeneration
The 4 step process of ion exchange chromatography
- Equilibration
- Sample application
- Elution
- Regeneration
Graph of the process
Y axis concentration of added NaCl
X axis elution volume
E
Sample injection volume, unbound molecules elute, elution of unwanted material, elution of target molecules, high salt wash, re-equilibration
4 sentences to describe the principle of ion exchange chromatography
Based on the ionic interaction of proteins and surface groups of beads.
Accessible surface charges on the protein compete to bind to functional groups of beads.
Proteins are sorted by anionic or cationic strength.
Proteins and surface groups on beads are oppositely charged.
Opposite charges attract
Cation exchange
Anion exchange
asymmetric charge distributions can allow binding despite net zero charge
4 interactions at the ion exchange
Functional groups on ion exchanger attract proteins of opposite charge.
Higher accessible surface charge on protein = better binding.
Sample proteins exchange with counter-ions of same charge bound to functional groups of exchanger during sample application.
Bound sample proteins then exchange with counter-ions (from elution buffer) of same charge to detach from functional groups of exchanger during elution.
Use cation or anion ion exchanger ?
At neutral pH: Blue protein is below its isoelectric point (thus is negatively charged while other red and green proteins are positively charged). Hence, use anion exchanger.
At neutral pH: Blue protein binds to an anion exchanger and can be separated from the other proteins which wash through. Hence, use cation exchanger
At neutral pH: Red and green proteins can be separated on a cation exchanger and
the blue protein washes through. Hence, use cation exchanger.
what beads do cation exchanger use
negatively charged beads
what beads do anion exchanger use
positively charged beads
Another thing to consider when deciding anion or cation besides pi of protein
One needs to consider stability (and functionality) of the protein under different pH when deciding to use either cation or anion exchanger.
What buffer ions should I use?
Use buffering ions that have the same charge as the ion exchanger functional group.
The buffer pKa value should be within 0.5 pH units of the desired working pH.
Elution of bound proteins, what ions should the buffer contain?
Ideally, addition of non-buffering salt such as NaCl to increase ionic strength
If you are purifying an enzyme, why cant the buffer pH be changed significantly?
Changes in pH may affect the shape of an enzyme so that either the substrate cannot bond to the active site or it cannot undergo catalysis.
Extreme pH can denature enzymes
6 steps involved in cation exchange chromatography using a salt gradient
The cation exchange column has a negatively charged surface.
It is initially bound by positively charged counter ions.
Proteins with a net positive charge bind to the surface of the cation exchange column.
The proteins displace the positively charged counter ions from the column.
Sodium chloride is added as a non-buffering salt, and positively charged sodium ions displace the bound proteins from the surface of the cation exchange column.
Negatively charged chloride ions which are co-ions to sodium ions, bind to the positively charged proteins and prevent them from binding to the cation exchange column.
6 steps involved in anion exchange chromatography using a salt gradient
The anion exchange column has a positively charged surface.
It is initially bound by negatively charged counter ions.
Proteins with a net negative charge bind to the surface of the anion exchange column.
The proteins displace the negatively charged counter ions from the column.
Sodium chloride is added as a non-buffering salt, and negatively charged chloride ions displace the bound proteins from the surface of the anion exchange column.
Positively charged sodium ions which are co-ions to chloride ions, bind to the negatively charged proteins and prevent them from binding to the anion exchange column
Linear gradient
Ionic strength of elution buffer is increased gradually.
Step gradient
Ionic strength of elution buffer is increased in discontinuous steps
2 advantages of stepwise elution over gradient elution
Stepwise elution has a faster separation time compared with gradient elution.
Stepwise elution results in reduced buffer consumption.
1 advantage of gradient elution over stepwise elution
Higher resolution of proteins with different binding affinities to the matrix.
2 factors that affect resolution during ion exchanger chromatography
Flow rate
Sample volume
How does flow rate affect resolution
The slower the flow rate, the greater the resolution
How does sample volume affect resolution
The smaller the sample volume, the greater the resolution
What buffering ions should we use?
Use buffering ions that have the same charge as the ion exchanger functional group.
