Reversed Phase Liquid Chromatography (RPC) has become an accepted tool for the separation of peptides, proteins and other biopolymers in the pharmaceutical, chemical and biochemical industries.
Reversed Phase Liquid Chromatography (RPC) separates molecules based on nonpolar interactions and requires a nonpolar stationary phase and a polar mobile phase. Typically the mobile phase consists of a mixture of water (buffer) and acetonitrile, methanol, THF, or 2-propanol.
Normal phase and hydrophilic interaction liquid chromatography (HILIC) are primarily used to separate polar and hydrophilic compounds. In reversed phase mode very polar compounds are often not sufficiently retained in low percent organic, or even in 100% aqueous mobile phase. The order of elution in normal phase is opposite that found in reversed phase for the same mixture of compounds. Although non-polar organic mobile phases and a silica stationary phase were used traditionally in normal phase LC, today most separations are performed with aqueous-organic mobile phases and a more polar-bonded stationary phase. This mode of HPLC is now commonly referred to as HILIC, hydrophilic interaction liquid chromatography.
By using an amide or amino bonded phase column, polar compounds can be retained by a normal phase or hydrophilic interaction chromatography retention mechanism. Typical mobile phases in HILIC are aqueous buffers with organic modifiers – primarily acetonitrile. In contrast to the retention behavior in reversed phase, in HILIC, solutes will be retained longer when increasing the percent acetonitrile.
Separation in Ion Exchange chromatography (IEC or IEX) is based on reversible adsorption of charged solute molecules to immobilized functional groups of opposite charge. Biomolecules generally have charged groups on their surfaces, which change with the buffer pH. Elution can be accomplished by changing the ionic strength or the pH, of which changing the ionic strength by increasing the salt concentration is most common.
IEX is further subdivided into cation exchange and anion exchange chromatography. Anion and cation exchange phases are classified as strong or weak, depending on how much the ionization state of the functional groups vary with pH. A strong ion exchange phase has the same charge density on its surface over a broad pH range, whereas the charge density of a weak ion exchange phase changes with pH, affecting its selectivity, which differs at different pH values.
