lecture 3 Flashcards
What are the 3 main stages to perform ion exchange chromatography?
- Equilibrium
- Sample application and wash
- Elution
What does the matrix contain for ion exchange chromatography?
matrix contains ionised groups
What happens when a proteins pI is above a pH?
It has an overall negative charge and will bind to a matrix that is positively charged
What is an anion exchanger?
itself is positive
- Binds anions
What is a cation exchanger?
- itself is negative
- Binds cations
what is the strength of the exchanger proportional to?
- proportional to the net charge
What is the matrix made of in ion exchange chromatography?
organic molecules
What is a weak anion exchanger?
Diethylamino ethyl (DEAE)
What is a strong anion exchanger?
Quaternary Ammonium (Q)
What is a weak cation exchanger ?
Carboxymethyl (CM)
What is a strong cation exchanger?
Methyl sulphonate (S)
What are the two key conditions needed for ion exchange chromatography?
- Low ionic strength buffer – bind
- pH to generate opposite charge to ion exchange resin – know your pI
What is chromatofocusing?
elution by changing pH
How does Chromatofocusing work?
- proteins with different pI separate as they pass through . molecules with the same isoelectric point are focused in narrow bands during the separation
How can elution by changing the salt occur?
- change the salt concentrations
- The salt ions compete with the protein for the charges on the stationary phase
- don’t change the pH
What types of molecules can elution by pH separate?
separate the protein isoforms
What are the advantages of ion exchange chromatography?
- Load large volume, elute small
- High capacity, good resolution
What are the disadvantages of ion exchange chromatography?
- Denaturation at pH extremes
- Product in high salt (tend to ppt)
- pH gradient technically difficult
How does hydrophobic interaction chromatography work?
separation via differing surface hydrophobicity , this is promoted by ‘salting out’
- salting out exposes the hydrophobic parts of the protein
How are proteins eluted in regards to hydrophobic interactions?
- elute in order of hydrophobicity
by interfering with the interaction ( decreasing salt, changing salt, pH , temp or detergent )
What is the drawback of elution by changing the salt concentration?
- may salt out on the column and not bind to the column at all
What are the parameters that need to be considered when using hydrophobicity?
- The ligand (: C4, C8 linear aliphatic chain, )
- Initial salt concentration
What type of technique is chromatofocusing?
Ion exchange chromatography
