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Principles of Chromatography

The physical characteristics of a molecule are exploited in liquid chromatography to affect a separation. The chromatographic modes listed below are defined based on the physical characteristics of the sample molecule:

Size Exclusion: This most basic separation mode is based on the size (hydrodynamic radius) of the molecule.

Ion Exchange: Anion or cation exchange chromatography is employed if a molecule in solution carries a positive or negative charge, as is the case with all proteins, peptides, nucleic acids and some carbohydrates.

Reversed Phase: This most common analytical HPLC mode is based on the interaction between non-polar regions in the molecule and a high density hydrophobic surface.

Hydrophobic Interaction: Like reversed phase, this mode is based on interaction between non-polar regions in the molecule but, unlike reversed phase, with a much lower density hydrophobic surface.

Affinity: The biological activity of a molecule is defined by its three dimensional structure. It is commonly used in chiral (for smaller molecules) or affinity (for larger molecules) chromatography.