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Principles of Chromatography

Principles of Chromatography 

The physical characteristics of a molecule are exploited in liquid chromatography to affect a separation. The most basic separation is based on the size (hydrodynamic diameter) of the molecule. When separating based on size, the chromatographic mode is size exclusion chromatography. 

If the molecule has a positive or negative charge associated with it, as is the case with all proteins, peptides, nucleic acids and some carbohydrates, cation exchange or anion exchange chromatography are employed. Hydrophobic regions on the molecule can be exploited by using normal phase, reversed phase, or hydrophobic interaction chromatography.

The three dimensional structure typically defines the activity of the molecule. Using this conformation of the molecule is very powerful separation technique and it is commonly used in chiral (for smaller molecules) or affinity (for larger molecules) chromatography. 

The following chart defines the chromatographic modes based on the physical characteristic used.

 

Physical Characteristics  Mode
Size (Hydrodynamic diameter)  Size Exclusion »
Molecular Charge  Ion (Anion, Cation) Exchange »
Mixed-Mode »
Hydrophobic Regions  Reversed Phase »
Hydrophobic Interaction »
Mixed-Mode »
Conformational Recognition  Affinity »
 Hydrophilic/Polar Regions HILIC »