Lecture 6 Gel Filtration Chromatography Flashcards
What is Ion Exchange Chromatography?
Components of a mixture are separated based on net charge of molecule, which interacts with the ion exchanger under the conditions of the experiment.
What does an ion exchange consist of?
Insoluble matrix chemically bound to charged groups and exchangeable counter ions
If matrix is positive, counter ion is negative, what is this?
Anion Exchanger
If matrix is negative, counter ion is positive, what is this?
Cation Exchanger
More highly charged molecules …
are tightly bound to resin -> travel slowly and eluted later
Moderately charged molecules…
Equilibrating between the resin->moving buffer more readily
Less charged molecules…
Bind less strongly to resin -> moving more readily and travels rapidly. Eluted sooner
What factors affects how the system can approach equilibrium?
- Rate of Irrigation (flow rate0
- Particle size of exchanger
- Ionic strength of irrigating solution
- Elution curve (perfect equilibrium, rarely reached in practice)
Name 3 Ion exchange media
- Ion exchange resin
- Cellulose ion exchangers
- Ion exchange gels
Describe Ion exchange media
- Cross linked polymer networks
- Hydrophobic
- High degree of substitution
- Low degree of permeability to macromolecules
- Sugars, nucleotides, amino acids
-E.g AAs on sulphated polystyrene resin (cation exchanger)
Describe Cellulose Ion Exchangers
- Open microstructure (advantage)
- Hydrophillic
- Degree of substitution = 1-2% of possible level
- Increased substitution leads to solubility in water.
- Covalent cross linkages can be used resulting in increased degree of substitution and higher capacity
Describe Ion exchange gels
- Cross linked dextran (Sephadex) or polyacrylamide (Biogel)
- Hydrophillic
- Minimal non-ionic adsorption
- Covalent cross linkages -> no solubility in water -> higher degree of substitution permitted
How do you remove contaminants in an experiment?
Washing - 6-8 HCl removes metal ions e.g iron.
Alcohol/ non ionic detergents removes lipid/proteins
How do you remove ‘fines’ (can reduce flow rate)?
Stir resin in water and allow to settle
Sephadex gels -> no fines
Degassing -> air/CO2 removed by vacuum
How do you equilibrate Cation exchange resin?
Washing HCl = H+ forms
How do you equilibrate Strong cation exchange resin?
Wash NaCl or NaOH = Na+ forms
How do you equilibrate Anion exchange resin?
Wash NaOH = OH- forms
How is cellulose equilibrated?
Wash with several volumes of starting buffer
How is DEAE (Diethylaminoethyl) Sephadex equilibrated?
supplied with Cl-
How is CM (Carboxymethyl) Sephadex equilibrated?
supplied with Na+
How long is swelling period
1-2 days at RT or 2hrs in boiling water bath
