Hydrophilic interaction liquid chromatography (HILIC) has become increasingly popular and promising for the separation of polar compounds, as HILIC allows class-based separation by hydrophilic interaction. Despite significant variations in chain length polarity, the presence of a similar hydrophilic headgroup in acyl-CoAs facilitates their elution within a relatively shorter time period. In HILIC chromatography, the compounds are separated on a polar stationary phase by gradually increasing solvent polarity . The compounds with higher polarity have enhanced affinity for the polar stationary phase, thus resulting in prolonged retention, whereas compounds with lower polarity tend to elute earlier.