Reverse-Phase Liquid Chromatography vs. Hydrophilic Interaction Liquid Chromatography

Liquid chromatography can be performed using various techniques, including reverse-phase and hydrophilic interaction liquid chromatography (HILIC). This method of chromatography involves an HPLC pump that pushes an analyte dissolved in a solvent (the mobile phase) under high pressure through a column (stationary phase) for separation. A detector and data system are then used to identify the compounds in the sample. HILIC columns are ideal for separating polar to highly polar compounds, which are often poorly retained in traditional reversed-phase LC separations. Modern, highly orthogonal HILIC phases, with short equilibration times and high efficiency, ensure reproducible and rapid chromatography results.

Reverse-Phase Liquid Chromatography

Reverse-phase chromatography is the most common type of liquid chromatography. In this method, alkyl or aromatic ligands are covalently bonded to the silica substrate, which provides a hydrophobic surface in the stationary phase where polar molecules pass through more quickly. One advantage of reversed phase chromatography is flexibility in the stationary phase. There are many different chemistries available for reverse phase chromatography applications, the most popular being an end-capped C8 or C18 phase.

Alternatives to the standard reversed phase columns are polar-embedded C8 and C18 bonded phases, which are compatible with 100% water and offer alternative selectivity. The combination of high carbon load, ultrapure silica, and silanol shielding provides superior peak shape and retention with highly polar samples. If polar embedded phases do not give your analytes enough retention, consider HILIC.

Hydrophilic Interaction Liquid Chromatography (HILIC)

In HILIC, organic solvents make up the weak mobile phase, while water is the strong eluting solvent. Because of this, HILIC has a more volatile and organic-rich mobile phase than reverse phase liquid chromatography. In return this gives HILIC an advantage with LC/ESI/MS as more efficient spraying and desolvation creates taller analyte peaks and reduces baseline noise for a more accurate analysis.

While Reverse-phase and HILIC chromatography methods differ in their elution order and retention of polar compounds, each holds advantages that make them an adequate chromatography testing method.