Choosing the Appropriate Separation Mode

Reversed-Phase (RP)

Use reversed-phase chromatography for hydrophobic and moderately polar compounds.

Typical applications include:

• Natural products
• Pharmaceuticals
• Cannabinoids
• Hydrophobic impurities
• Environmental contaminants

Examples

• Cannabinol
• Limonin

          

Increasing Retention in RP

To increase retention:

• Increase the water content of the mobile phase.
• Reduce the percentage of organic solvent.

HILIC - Mixed Mode

Use HILIC when compounds are too polar for effective retention in reversed-phase chromatography.

Typical applications include:

• Polar pharmaceuticals
• Organic acids
• Amines
• Metabolites
• Highly water-soluble compounds

Examples

• Metformin
• Methotrexate

Increasing Retention in HILIC

To increase retention:

• Increase the organic content of the mobile phase.
• Reduce the aqueous content.

Before Using the Column

Before installing a new column:

1. Completely purge instrument solvent lines of previous mobile phases.
2. Purge the injector to remove residual sample contamination.
3. Confirm all solvents are fresh, HPLC-grade, and properly degassed.
4. Prepare fresh buffers daily.
5. Verify all tubing and fittings are clean and properly connected.

Initial Equilibration

1. Install the column following standard laboratory procedures.
2. Equilibrate with a 50:50 organic solvent/water mixture containing all planned mobile-phase additives.
3. Continue equilibration for approximately 30 minutes.
4. After equilibration, switch to your initial method conditions and allow the system to stabilize.

Recommended Protection

For dirty matrices or challenging samples:

• Use inline filters whenever possible.
• Use guard columns whenever possible.
• Replace protection devices routinely as part of preventive maintenance.

Acidic Compounds

To maximize reversed-phase retention:

• Acidify the mobile phase with approximately 0.1% formic acid.

This suppresses ionization and increases hydrophobic retention.  Method development can then follow the same approach used for neutral compounds.

Basic Compounds

Many hydrophobic bases can be analyzed successfully by RP chromatography.

However:

• Small highly polar bases may show limited RP retention.
• These compounds often perform better in HILIC.

Avoid operating at unnecessarily high pH values that may shorten column or instrument lifetime.
 

Troubleshooting Common Problems

Common causes of poor chromatographic performance include:

• Inadequate equilibration
• Contaminated solvent lines
• Improper sample solvent
• Dirty mobile phases
• Worn fittings
• Buffer precipitation
• Insufficient filtration

Related Resources

• View TYPE-C™ Column Specifications and Troubleshooting Guidelines
• View TYPE-C™ HPLC Column Troubleshooting Tips

Storage Instructions

At the end of use:

1. Flush the column with a mobile phase containing approximately 90:10 organic solvent/water.
2. Remove all system pressure before disconnecting the column.
3. Store under recommended solvent conditions.

Important: Never disconnect a column while pressure remains in the system. Pressure shock can damage column hardware and stationary phase performance.

Related Resources

• View Complete TYPE-C™ HPLC Column Storage Instructions

Best Practices Summary

• Use HPLC-grade solvents only.
• Degas all mobile phases.
• Replace buffers daily.
• Equilibrate thoroughly before analysis.
• Use column filters and guard columns whenever possible.
• Increase water to increase RP retention.
• Increase organic solvent to increase HILIC retention.
• Store columns in approximately 90:10 organic/water solvent.