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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. 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.

Applications

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.

 

The silica-based TSKgel® reversed phase column line offers a variety of stationary phased designed for the analysis of either large molecules, such as peptides, proteins and oligonucleotides, or low molar mass analytes, such as:

  • pharmaceutical drugs
  • forensic compounds
  • derivatized amino acids
  • carbohydrates
  • steroids, lipids, fatty acids

TSKgel® silica-based reversed phase columns are grouped into 6 product families:

    TSKgel® Column Type

    Particle Size
    (µm)

    Benefit
    Monomeric Bonded Silica (8 nm) 5, 10 General purpose columns; 8 nm pores allows fast mass transfer of solutes in the 100-6,000 Da molar mass range
    ODS 100V and 100Z (10 nm) 3, 5 1st choice for general purpose columns; two levels of hydrophobicity: 15% (V), 20% (Z)
    High Efficiency (14 nm) 2 Superior resolution and speed; improved sensitivity; savings in time and solvent usage
    Specialty Silica Columns 5, 10 Provides optimum separation for oligonucleotides, polyaromatic hydrocarbons, synthetic peptides
    ODS-140HTP for High Throughput 2.3
    High throughput separations; high resolution and short analysis time at moderate pressures
    TMS-250 and Protein C4-300  3, 10 Ideal for separation of large biomolecules, such as proteins

    The polymethacrylate-based TSKgel® reversed phase columns are available in a range of pore and particle sizes. Key advantages of these polymer-based columns include:

    • Chemically stable from pH 2 to 12, allowing many basic compounds to be analyzed in their uncharged form, thus reducing secondary adsorption and improving peak shape

    • Improved recovery for peptides and proteins due to reduced secondary interactions

    The TSKgel® polymer-based reversed phase columns are an excellent choice for large molecular weight (MW) biomolecules (>10,000 Da) and for analyzing small MW compounds at high pH and are offered in 4 different chemistries.