What is chromatography?

Chromatography

Technique used for the separation of mixtures. The mixture is carried through a stationary phase by the mobile phase

Stationary phase = the resin

Mobile phase = the eluent (mostly water)

The separation is based on physico-chemical properties of the different components generating differences of affinity between the phases

Separation mechanisms principles

1. Size exclusion chromatography

2. Affinity chromatography

3. Ion exclusion chromatography

4. Acid retardation chromatography

1)Size exclusion chromatography

Separation based on size of the molecules to purify

- Molecular sieving

- Porosity of the resin to be adapted to molecular weight

- Works with strong cation exchangers (monovalent Na+, K+)

Very small molecules enter many pores, last to elute

Large molecules enter few pores, first to elute.

2)Affinity chromatography

- Separation based on specific interactions with the resin

- Ligand exchange

- Interaction between the resin and the hydroxyl groups (OH) of the sugar molecules

- Affinity depends on the spatial configuration of the molecule

- Works with strong cation exchangers (Ca2+, Pb2+)

3)Ion exclusion chromatography

- Separation based on charge

- Works with strong cation exchangers (monovalent form: Na, K)

Neutral Molecules enters the pores, last to elute

Ions are rejected by electric repulsion, first to elute.

4)Acid retardation chromatography

- Separation based on affinity and difference of ionization

- Works with weak base resin (SO42- form)

Protonated organic acids (neutral) are retained by affinity and not excluded by ions, last to elute

Other impurities have less affinity than organic acids and diffuse in the resin, second to elute

Ions are rejected by electric repulsion, first to elute.