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HPLC Column Selection Guide

Choice of column is important as there are 3 distinct separation modes, each with their unique characteristics.

HPLC Separation Modes

Liquid Chromatography uses liquid as an eluent, also known as mobile phase. It is an analytical method that separates a mixture of compounds based on their physical and chemical differences. High Performance Liquid Chromatography (HPLC) is a method that introduces the mobile phase under high pressure conditions, resulting in rapid and high-performance separations. The various interactions between the analyte, packing material of the analytical column (stationary phase) and mobile phase are key factors for successful separation. There are a wide variety of separation modes, which can be achieved by using specific combinations of both stationary and mobile phases.

Reverse Phase Chromatography (RPC)

  • Separation is based on the partition equilibration between stationary phase and mobile phase
  • The polarity of the stationary phase is lower than that of the mobile phase
  • Using a lower polarity mobile phase makes elution of target compounds quicker
  • The mobile phase typically contains a mixture of organic solvents (methanol, acetonitrile or THF) and aqueous solvents (water or buffer)

Normal Phase Chromatography (NPC)

  • Separation is based on the partition equilibration between the stationary phase and mobile phase
  • The polarity of the stationary phase is higher than that of the mobile phase
  • The mobile phase typically contains a mixture of organic solvents with different polarities such as hexane and isopropanol

Hydrophilic Interaction Chromatography (HILIC)

  • Separation is based on hydrophilic interaction
  • A high polarity stationary phase is used
  • The mobile phase typically contains a mixture of organic solvents (acetonitrile0 and aqueous solvents (water or buffer)
  • Recommended for the analysis of high polar compounds

 

When it comes to RPC, the stationary phase is usually made of silica base material to which C8 or C18 alkanes are bound. For NPC and HILIC different phases or functional groups could be attached, more polar compared to the C18 and C8 from the RPC. Also, different base material could be found in the column such as polyvinyl alcohol, polyhydroxy methacrylate, styrene-divinylbenzene copolymer, polyvinyl alcohol or polymethacrylate. However, the choice of the right column is based on the application and targeted compounds as well.

Refer to our HPLC Column Selection Guide for advice.

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