Why does null hypothesis testing matter for glycan separation validation?

Null hypothesis testing ensures that observed differences in glycan retention times or peak intensities are statistically significant, supporting confident target validation and reducing false positives in glycan profiling.

How does independent variable isolation fit HILIC glycan analysis?

Isolating variables such as mobile phase composition and flow rate allows teams to attribute changes in glycan separation directly to experimental conditions, strengthening discovery-stage optimization and reproducibility.

What do quantitative dependent variable measurements enable in HILIC workflows?

Quantitative measurements of fluorescence intensity and retention time enable precise comparison of glycan species, facilitating robust assay development and supporting data-driven advancement decisions.

Why are replication requirements critical for cross-functional glycan analytics?

Replication ensures that glycan separation and detection are consistent across runs and teams, enabling reliable data sharing and cross-functional collaboration in biologics development.

What statistical analysis capabilities are required before HILIC implementation?

Teams must apply statistical tools to assess chromatogram reproducibility, peak resolution, and signal-to-noise ratios, ensuring analytical robustness before integrating HILIC into broader R&D workflows.

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

JoVE Encyclopedia of Experiments

Biological Techniques

0 views • 3:07 min • July 8th, 2025

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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.

First, open the software to control the mobile phases. Wash UPLC instruments with 50% solvent B and 50% solvent C at the flow rate of 0.2 milliliters per minute, for 30 minutes. Then wash, with 25% solvent A and 75% solvent B at a flow rate of 0.2 milliliters per minute, for 20 minutes, then, at a flow rate of 0.4 milliliters per minute.

Dissolve the labeled N-glycans with 25 microliters of a mixture of 100% acetonitrile and ultra-pure water at a 2:1 (volume/volume) ratio. Then, centrifuge at 134 x g for 5 minutes at 4 degrees Celsius, and use a drive pipe to load 10 microliters of the supernatant into the UPLC instruments.

Separate the labeled N-glycans at a flow rate of 0.4 milliliters per minute, with a linear gradient of 75% to 62% acetonitrile for 25 minutes. Then, perform an analytical run by Dextran Calibration Ladder/Glycopeptide column on a UPLC at 60 degrees Celsius.

Detect N-glycan fluorescence at excitation and emission wavelengths of 330 nanometers and 420 nanometers respectively.