Hydrophilic Interaction Liquid Chromatography (HILIC) has evolved into a powerful chromatographic technique for the retention and separation of hydrophilic neutral and polar compounds, which are often difficult to retain by Reversed-Phase Liquid Chromatography (RPLC). HILIC utilises a polar stationary phase combined with an organic/aqueous mobile phase [1], typically containing a high percentage of the organic component (~>60% acetonitrile). In HILIC, water is the strong solvent and unlike RPLC, increasing the percentage of water in the mobile phase decreases analyte retention (Figure 1).
Mechanistically HILIC is complex, involving a combination of multiple modes of interaction between the analyte, stationary phase and eluent. In HILIC, a water-enriched layer is present at the stationary phase surface, as shown in Figure 2. A minimum of ~3% water is required in the mobile phase for formation of this layer. Analyte retention is often due to a combination of partitioning of the analyte into the water-enriched layer, together with hydrogen bonding and electrostatic interactions [2-5].
When to consider HILIC
HILIC is applicable to hydrophilic analytes that are challenging to retain by RPLC. Analyte log P and log D values can provide an indication of the suitability of the analyte for retention by HILIC. Generally, if an analyte elutes before caffeine in RPLC (log P ~zero), it may be suitable for analysis by HILIC, whilst those eluting later may be better suited for RPLC.
FIGURE 1. Solvent elution strength in HILIC mode.>> Download the full Application Note as PDF
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