Overview

One of the unique characteristics of Cogent™ TYPE-C™ silica hydride columns is their ability to support multiple retention mechanisms. Depending on the column chemistry, analyte properties, and mobile phase composition, both HILIC and reversed phase interactions may contribute to retention.

This flexibility can provide unique selectivity for complex samples and mixed analyte classes.

However, it is uncommon for both retention mechanisms to contribute equally to the separation of a given compound under the same chromatographic conditions.

Can HILIC and Reversed Phase Retention Occur at the Same Time?

Yes.

On TYPE-C™ columns, it is possible for both HILIC and reversed phase interactions to influence analyte retention simultaneously.

This is especially true for:

• Amphiphilic compounds
• Molecules containing both polar and non-polar regions
• Metabolites
• Pharmaceuticals
• Peptides
• Complex biological analytes

In these cases, retention may result from a combination of:

• Polar interactions
• Hydrophobic interactions
• Surface adsorption effects
• Other analyte-specific interactions

Why Equal Contributions Are Uncommon

Although both mechanisms may be present, they rarely contribute equally.

For most compounds:

• One retention mechanism becomes dominant.
• The other plays a secondary role.
• The balance depends on the analyte structure and mobile phase composition.

Two different compounds analyzed under the same method conditions may even exhibit different dominant retention mechanisms.

For example:

• One analyte may be retained primarily through HILIC interactions.
• Another may be retained primarily through reversed phase interactions.

This is one reason TYPE-C™ columns often provide unique selectivity and elution order compared to traditional stationary phases.

Mobile Phase Composition Controls Retention Balance

The relative importance of HILIC and reversed phase retention can be adjusted through mobile phase composition.

High Organic Conditions

When using:

• High acetonitrile content
• Low aqueous content

HILIC retention generally becomes more significant for polar compounds.

Higher Aqueous Conditions

As water content increases:

• Reversed phase interactions often become more prominent.
• Hydrophobic retention may increase for suitable analytes.

Because of this, the dominant retention mechanism can change as mobile phase composition changes.

Effects During Gradient Methods

Gradient methods can shift the balance between retention mechanisms throughout the separation.

As solvent composition changes:

• HILIC contributions may increase or decrease.
• Reversed phase contributions may increase or decrease.
• Selectivity can change.
• Elution order may shift.

This flexibility is a valuable tool during method development.

Method Development Opportunities

The ability to manipulate multiple retention mechanisms allows chromatographers to explore separations that may not be achievable using:

• Conventional reversed phase columns
• Traditional HILIC columns
• Normal phase columns

Adjustments to:

• Organic solvent content
• Water content
• Additives
• Gradient profiles

can significantly influence how analytes interact with the stationary phase.

Applications Where Mixed Retention Is Useful

Simultaneous HILIC and reversed phase contributions can be beneficial when analyzing:

• Metabolites
• Pharmaceuticals
• Biological samples
• Polar impurities
• Complex mixtures
• Amphiphilic compounds

These analytes often possess both hydrophobic and hydrophilic regions, making them well suited to the unique selectivity available from TYPE-C™ columns.

Key Takeaways

• HILIC and reversed phase retention can occur simultaneously on TYPE-C™ columns.
• Equal contributions from both mechanisms are uncommon.
• One retention mechanism is typically dominant under a given set of conditions.
• Mobile phase composition strongly influences the balance between HILIC and reversed phase retention.
• Gradient methods can change the relative contribution of each mechanism during a run.
• This mixed retention behavior is one reason TYPE-C™ columns provide unique chromatographic selectivity.