HILIC can be described as a variation of reversed phase chromatography performed using a polar stationary phase. The mobile phase employed is highly organic in nature (> 60-70% solvent, typically acetonitrile) containing a small percentage of aqueous solvent/buffer or other polar solvent. The water in the mobile phase forms an aqueous-rich layer adsorbed to the polar surface of the stationary phase into which analytes partition.

The primary advantage of HILIC as a separation technique is the strong retention of polar, hydrophilic compounds that are unretained under conventional reversed phase conditions, without the requirement for an ion-pair additive in the mobile phase.