Reversed Phase Chromatography
Starting in the mid 1970s Reversed Phase Liquid Chromatography (RPC) has become the standard technique to analyze small molecular weight compounds. More recently, RPC has become an accepted tool for the separation of peptides, proteins and other biopolymers in the pharmaceutical, chemical and biochemical industries.
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.
RPC can be applied to the analysis of a wide variety of compounds, ranging from neutral polar and nonpolar solutes to acidic, basic, and amphoteric compounds. RPC is also an efficient technique for the analysis of derivatized amino acids, peptides and proteins, although protein structure is not always maintained due to the high concentration of organic solvent required for their elution.
TSKgel Reversed Phase Columns
Silica particles are most commonly used as the support in TSKgel reversed phase columns, which is then derivatized with octadecylsilane (ODS, C18) or smaller hydrocarbon chains such as C1 (Methyl), C4 (Butyl), C5 (Phenyl), or C8 (Octyl).
Polymer-based supports have been introduced as an alternative to TSKgel silica-based reversed phase columns, particularly for analyzing basic compounds in their neutral state at high pH.