Techniques
Five separation modes: how each one works, when to use it, and what the trace can and cannot tell you.
Size exclusion (SEC)
Separate by hydrodynamic behaviour and pore access. Isocratic, no binding step.
Ion exchange (IEX)
Separate by surface charge. High resolution and scalable; common for polishing.
Hydrophobic interaction (HIC)
Bind at high salt, elute on a decreasing salt gradient by surface hydrophobicity.
Affinity
Capture through a tag or epitope: His-tag, GST, MBP, protein A/G for antibodies.
Reversed phase (RPC)
Organic-solvent gradients for peptides and small molecules. High resolution, usually denaturing.
