Reversed Phase Chromatography

Reversed Phase Chromatography

Starting in the mid 1970s reversed phase liquid chromatography (RPC) has become the standard technique to analyze small molecular weight compounds in industrial, academic and governmental laboratories. More recently, RPC has become the accepted tool for the separation of peptides, proteins and other biopolymers in the pharmaceutical, chemical and biochemical industries.

The opposite of normal phase chromatography, RPC requires a non-polar 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.

Silica particles are most commonly used as the support, which then is derivatized with octadecylsilane (ODS). Polymer-based supports have been introduced as an alternative to silica-based reversed phase columns, particularly for analyzing basic compounds in their neutral state at high pH.

RPC columns can be applied to the analysis of a wide variety of compounds, ranging from neutral polar and non-polar 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.

Tosoh Bioscience offers 14 distinct Reversed Phase column types which are either based on silica or methacrylate particles.


The silica-based TSKgel reversed phase column products consist of 12 stationary phases over 6 key product families. Each TSKgel silica column features:

  • High purity Type B Silica
  • HIgh efficiencies 
  • Excellent recoveries
  • Low bleed for MS
An excellent choice for analysis of small molecules, peptides and proteins. TSKgel silica-based reversed phase columns are grouped into 6 product families:

The polymeric-based TSKgel reversed phase columns are synthesized from polymethacrylate polymers in a range of pore and particle sizes. Advantages of polymethacrylate particles include:

  • Hydrophilic backbone to improve recovery and reduce secondary interactions
  • pH stable from 1 to 12
  • Compatibility with organic solvents eliminates swelling

An excellent choice for large MW biomolecules (>10,000 Da) and for analyzing small MW compounds at high pH. TSKgel polymeric reversed phase columns are offered in 4 different chemistries: