Updated 5-JUNE-2025

Improving Polar Compound Analysis: ANP vs. HILIC for LC and LC-MS

Over the years, analytical scientists have faced persistent challenges when developing HPLC and LC-MS methods for highly polar compounds. While Hydrophilic Interaction Liquid Chromatography (HILIC) has been a go-to technique, many chromatographers report significant drawbacks—especially when using gradient methods.

Common HILIC Challenges

• Slow Equilibration: HILIC columns, like conventional silica-based phases, require extended equilibration due to the persistent water layer (hydration shell) on the silica surface. This leads to increased downtime between runs.
• Reproducibility Issues: The hydration shell is sensitive to environmental changes (e.g., temperature), often resulting in poor precision and retention time variability.
• Column Instability: HILIC columns may degrade unpredictably, particularly during overnight sequences.
• High Salt Requirements: Retention often depends on mobile phases with salt concentrations up to 100 mM—problematic for LC-MS systems and preparative workflows due to ion suppression and fouling.

Aqueous Normal Phase (ANP): A Smarter Alternative

ANP chromatography, particularly with Cogent TYPE-C™ silica hydride columns, offers a robust solution. Like HILIC, ANP retains polar compounds using high-organic mobile phases and inverse gradients. However, the underlying chemistry is fundamentally different:

• No Hydration Shell: TYPE-C silica lacks the persistent water layer, enabling faster equilibration (typically 3–5 column volumes).
• Minimal Silanol Activity: With <5% residual silanols and direct Si–C ligand bonding, ANP columns avoid the secondary interactions that plague HILIC.
• Low Salt Operation: ANP methods typically require ≤15 mM salt, eliminating the need for buffers and reducing LC-MS contamination risk.

Key Benefits of ANP over HILIC

• Rapid Equilibration → Higher throughput and reduced solvent use.
• Exceptional Precision → Reliable retention times across runs and days.
• Extended Column Lifetime → Fewer failures and lower replacement costs.
• LC-MS Compatibility → Low salt minimizes ion suppression and system fouling.
• Versatile Selectivity → Retains both polar and some non-polar analytes.
• Simplified Method Development → Broad selectivity range and reproducibility.

Technical Highlights

1. Surface Chemistry: Cogent TYPE-C columns exhibit <1 monolayer of water vs. 3–12 in HILIC, improving surface consistency.
2. Fast Re-equilibration: 3–5 column volumes vs. significantly longer for HILIC.
3. Stable Surface Charge: Silica hydride surfaces are negatively charged due to surface hydroxide ions.
4. Dual-Mode Capability: TYPE-C columns can operate in both ANP and reversed-phase modes.
5. Low Additive Requirements: Most polar analytes can be retained with ≤15 mM salt—no buffers needed.

For further reading, see:

Kulsing et al., "Insights into the Origin of the Separation Selectivity with Silica Hydride Adsorbents," J. Phys. Chem. B, 2015, 119, 3063–3069.