Hydrophilic Interaction Liquid Chromatography: A Technique to Separate Hydrophilic Polar Analytes Using Hydrophilic Beads

Hydrophilic interaction liquid chromatography, HILIC, is a technique to separate hydrophilic polar compounds based on their degree of polarity. Begin by setting up a HILIC column packed with hydrophilic beads carrying polar functional groups.

First, condition the column using an equilibration buffer and mobile phase - a mixture of organic solvent and water. The buffer molecules stabilize the pH, while organic solvent saturates the column. The water molecules immobilize and create an aqueous layer over the hydrophilic beads.

Load the sample containing a mixture of glycans - hydrophilic polar compounds - labeled with a fluorescent tag. Run the sample using a mobile phase gradient through the column at an optimum flow rate.

As the sample moves along the hydrophobic mobile phase, the glycans migrate into the aqueous layer, leaving behind the impurities. Subsequently, glycans move toward the polar functional groups on the beads. Here, more polar glycans bind strongly, while less polar ones bind loosely.

As the mobile phase hydrophobicity decreases, weakly bound glycans with less polarity and a shorter retention time elute first. Conversely, strongly bound glycans with higher polarity and a longer retention time elute last.

Finally, a detector detects the fluorescent signal of the eluted fractions, corresponding to standard glycan peaks in the chromatogram.