Introduction

One of the most common challenges in HPLC is obtaining sufficient retention for highly polar compounds. Many acids, bases, amino acids, metabolites, peptides, carbohydrates, and other hydrophilic molecules elute near the solvent front when analyzed using conventional reversed phase methods.

HILIC (Hydrophilic Interaction Liquid Chromatography) has become a widely accepted chromatographic approach for analyzing these compounds. Cogent™ TYPE-C™ silica hydride columns offer unique HILIC capabilities that provide strong retention of polar analytes while maintaining excellent compatibility with LC-MS detection.

Historically, MICROSOLV referred to this retention behavior as Aqueous Normal Phase (ANP). Today, the industry-recognized term HILIC is used to describe these polar compound separations performed under organic-rich mobile phase conditions.

What Is HILIC?

HILIC is an HPLC mode used to retain and separate polar compounds using mobile phases that contain a high percentage of organic solvent, typically acetonitrile, along with smaller amounts of water and volatile additives.

Typical HILIC mobile phases include:

• Acetonitrile
• Water
• Formic acid
• Acetic acid
• Ammonium formate
• Ammonium acetate

These conditions are particularly useful for compounds that show little retention in reversed phase chromatography.

Why Use HILIC?

HILIC is often selected when analyzing:

• Organic acids
• Amines
• Polar pharmaceuticals
• Metabolites
• Peptides
• Carbohydrates
• Nucleotides
• Water-soluble compounds

Benefits include:

• Enhanced retention of polar compounds
• Excellent LC-MS compatibility
• Improved separation selectivity
• Strong signal response in mass spectrometry
• Reduced dependence on ion-pairing reagents

HILIC with TYPE-C™ Columns

All Cogent™ TYPE-C™ columns can operate under HILIC conditions, including:

• Diamond Hydride™
• Phenyl Hydride™
• Amide™
• Silica-C™

Each phase provides its own selectivity while utilizing the unique silica hydride surface chemistry characteristic of TYPE-C™ technology.

Rather than behaving exactly like conventional HILIC columns, TYPE-C™ columns provide a distinctive retention profile that can be highly useful for challenging polar compound separations.

This often allows chromatographers to achieve separations that may be difficult using either traditional reversed phase or conventional HILIC stationary phases.

Typical HILIC Mobile Phases

Many HILIC methods on TYPE-C™ columns begin with high levels of organic solvent.

Typical starting conditions may include:

• 95% acetonitrile / 5% aqueous
• 90% acetonitrile / 10% aqueous
• 80% acetonitrile / 20% aqueous

The precise composition depends on the analytes being studied.

As water content increases, retention generally decreases and compounds elute from the column.

Advantages of TYPE-C™ Columns for Polar Compounds

When used under HILIC conditions, TYPE-C™ columns offer several practical benefits:

• Strong retention of highly polar compounds
• Fast equilibration between runs
• Excellent reproducibility
• Unique selectivity
• Broad method development flexibility
• Excellent LC-MS performance

These characteristics have made TYPE-C™ columns popular for analytical, bioanalytical, environmental, pharmaceutical, food, and metabolomics applications.

Flexible Method Development

A unique feature of TYPE-C™ columns is their ability to operate in multiple chromatographic modes.

Depending on mobile phase composition, many TYPE-C™ columns can be used in:

• HILIC mode
• Reversed Phase mode
• Normal Phase mode

This flexibility allows method developers to explore different retention mechanisms without changing columns.

Some phases may be more optimized for polar analytes, while others may provide stronger performance for mixed-mode or reversed phase applications.

HILIC Versus Reversed Phase

Reversed Phase

Best suited for:

• Non-polar compounds
• Moderately polar compounds
• Hydrophobic analytes

Typical mobile phases contain:

• High water content
• Lower organic content

HILIC

Best suited for:

• Polar compounds
• Hydrophilic analytes
• Metabolites
• Charged compounds

Typical mobile phases contain:

• High organic content
• Lower water content

Many compounds that elute too quickly in reversed phase chromatography can often be retained effectively in HILIC mode.

Key Takeaways

• HILIC is widely used for the analysis of polar compounds.
• TYPE-C™ columns provide unique HILIC capabilities for challenging analytes.
• Diamond Hydride™, Phenyl Hydride™, Amide™, and Silica-C™ columns can all be used under HILIC conditions.
• High-organic mobile phases provide strong retention of many polar compounds.
• HILIC methods are highly compatible with LC-MS.
• TYPE-C™ columns offer fast equilibration, reproducibility, and unique selectivity.