Managing the column equilibration time in hydrophilic interaction chromatography.

Area of Science:

• Analytical Chemistry
• Chromatography

Background:

• Hydrophilic interaction chromatography (HILIC) is crucial for separating polar compounds.
• Achieving consistent and rapid column equilibration is essential for reproducible HILIC separations, especially in gradient elution.

Purpose of the Study:

• To investigate the feasibility and efficiency of partial column equilibration in HILIC gradient elution.
• To determine the optimal conditions for rapid HILIC method setup and analysis.

Main Methods:

• Gradient elution was employed with various hydrophilic interaction chromatography stationary phases.
• Partial column equilibration was assessed by purging with mobile phase volumes as low as 12 column volumes.
• Retention time reproducibility was evaluated using relative standard deviations after conditioning runs.

Main Results:

• Repeatable partial equilibration was achieved for diverse HILIC stationary phases using ~12 column volumes.
• Excellent retention time reproducibility (RSD ~0.15%) was obtained after 1-2 conditioning runs.
• Partial equilibration was largely independent of stationary phase type and gradient slope, though constant equilibration times are critical.

Conclusions:

• Partial column equilibration offers a time-efficient alternative to full equilibration for many HILIC applications.
• Optimized conditions, including temperature and mobile phase volume, can facilitate rapid method setup and analysis.
• Modern instrumentation accommodates the strict control of equilibration periods needed for selectivity.