HPLC Column Selection Guide

Choice of column is important as there are 3 distinct separation modes, each with their unique characteristics, such as reverse phase chromatography (RPC), normal phase chromatography (NPC) and hydrophilic interaction chromatography (HILIC). RPC separation is based on the partition equilibration between the stationary phase and mobile phase of the analyte. The polarity of the stationary phase is lower compared to the mobile phase. In other words, the stationary phase of the column is nonpolar and the analytes to be separated have a low polarity. A lower polarity of the mobile phase decreases the analysis time, and its composition is made of 2 distinct parts, an aqueous part and an organic part. On the other side, NPC presents the same separation mechanism as RPC, but the polarity of the stationary phase is higher compared to the mobile phase. Lastly, HILIC presents a different separation mode which focuses on the hydrophilic interactions between the stationary phase and targeted compounds. The stationary phase is highly polar, and for mobile phase buffers are usually involved. HILIC is used 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.

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