Flashcards in lecture 3 Deck (89)
Chromatography can be applied to which elements within chasm
4- Molecular weight
Ion exchange theory works on the basis of?
Overall charge can be determined if we know the sequence
Surface charge vs overall protein charge?
may be different
What type of interaction is occurring in ion exchange chromatography?
Matrix within Ion exchange chromatography contains
Anion exchange therefore?
cats pur positive
Strength of the interaction between the ionised groups and the protein is due to?
number of ionic charges the proteins have
separation due to charge
Matrix functional group examples
- Diethylamino ethyl (DEAE)
- Quaternary ammonium
- Methyl sulphonate
Stronger anions and cations
will interact over a larger range
issue with really strong or really weak binders?
will bind too much or not enough
Issue with anion and cation exchanges
may not be the appropriate pH
Conditions for adsorption?
Low ionic strength buffer
pH to generate opposite charge to ion exchange resin (know pI)
protonated (positively charged)
pI for a cation exchanger
- high (neg can bind to positive)
How to choose buffer pH
one pH unit away from the pI
Choose a suitable buffer pH for a cation exchanger when the pI=8.5
Can choose 1 pH unit above or bellow
- 9.5- too high (alkali)
- 7.5 better due to this being a cation exchanger (therefore your ions will be pos charged)
Elution method ?
change in ionic strength
Highly charged ions will come off?- elution
What type of gradient is drown in elution
What needs to be maintained during elution
pH as this may change the proteins ability to bind
Choose gradient such that?
they ate separated
Elution by changing the pH
Molecules with the same isoelectric point are focused in narrow bands during the separation.
What type of gradient in the collum- Elution by changing the pH. Usually is ?
usually low to high
when might we want to use the focussing technique to separates proteins?- range ?
usually over a range of 1 and a Half pH unit
Advantages of ion exchange chromatography?
1- Load large volumes, elute small (concentrating)
2- High capacity, good resolution
Disadvantages of ion exchange chromatography?
denaturation at extreme pH
product in high salt
pH gradient technically difficult to produce
Seperation due to?- Hydrophobic interaction chromatography
differing surface hydrophobicity