The Cogent Bidentate C8™ HPLC Column is a TYPE-C™ silica hydride stationary phase that provides excellent performance in both reversed-phase (RP) and HILIC chromatography. Compared to Bidentate C18™, the C8 phase generally provides lower hydrophobic retention, making it particularly useful when C18 retention is excessive or when faster elution and shorter run times are desired.
This guide provides recommended startup procedures, method development strategies, storage instructions, and best practices to help achieve optimal chromatographic performance and long column life.
Related Resources
- View Cogent Bidentate C8 HPLC Column Ordering Information
- View General Instructions for All Cogent TYPE-C HPLC Columns
Choosing the Appropriate Separation Mode
Reversed-Phase (RP)
Use Bidentate C8 for hydrophobic and moderately polar compounds where:
- C18 retention is excessive
- Faster analysis times are desired
- Reduced hydrophobic retention is beneficial
- Alternative selectivity to C18 is needed
Typical Applications
- Pharmaceuticals
- Drug impurities
- Natural products
- Environmental compounds
- Routine analytical methods
Example Compounds (Structures Shown Below)
- Tramadol
- Guanfacine
Increasing Retention in RP
- Increase water content.
- Reduce organic solvent content.
HILIC - Mixed Mode Chromatography
Use HILIC when compounds are too polar for effective reversed-phase retention.
Typical Applications
- Amines
- Polar pharmaceuticals
- Metabolites
- Organic acids
- Highly water-soluble compounds
Example Compounds (Structures Shown Below)
- Metformin
- Methotrexate
Increasing Retention in HILIC
To increase retention:
- Increase organic solvent content.
- Reduce aqueous content.
Before Using the Column
Before installation:
- Purge all solvent lines of previous mobile phases.
- Purge the injector system.
- Verify that all solvents are fresh and HPLC-grade.
- Degas solvents prior to use.
- Prepare fresh buffers daily.
Startup Instructions
Initial Conditioning
- Install the column using standard laboratory procedures.
- Verify all fittings and tubing are properly connected.
- Condition the column using a 50:50 organic solvent/water mixture that contains all intended method additives.
- Equilibrate for approximately 30 minutes.
- Switch to the initial mobile-phase conditions for the method and allow stabilization.
Column Protection
When analyzing challenging matrices:
- Use inline filters whenever possible.
- Use guard columns whenever possible.
- Replace filters routinely.
Reversed-Phase Method Development
Neutral Compounds
Recommended starting mobile phases:
- Acetonitrile / Water
- Methanol / Water
For most applications:
- Add 0.1% formic acid to both mobile-phase components.
This is especially important for LCMS methods.
Simple Samples
Start with:
- 70-90% organic solvent
Increase water gradually until desired retention is achieved.
Complex Samples
Suggested screening gradient:
- 90% organic
- To 10% organic
- Over approximately 10 minutes
Adjust composition and gradient slope as needed.
Acidic Compounds
To maximize retention:
- Use approximately 0.1% formic acid to suppress ionization.
Method development can then follow the same approaches used for neutral compounds.
Basic Compounds
Many hydrophobic bases retain well in reversed-phase mode.
However:
- Smaller highly polar bases may not retain adequately.
- These compounds often perform better in HILIC mode.
Operation at unnecessarily high pH is generally not recommended.
HILIC Method Development
Polar Neutral Compounds
Only sufficiently polar neutral compounds are likely to show meaningful HILIC retention. The same development strategies used for acids can often be applied successfully
Acidic Compounds
To maximize retention:
- Ensure the analyte remains ionized.
Recommended starting buffer:
- 10 mM ammonium formate
- or
- 10 mM ammonium acetate
Recommended starting pH:
- Approximately 6.5
Simple Samples
Begin with:
- 50% aqueous buffer
- 50% acetonitrile
Increase organic content as retention requires.
Complex Samples
Suggested scouting gradient:
- 90% acetonitrile
- To 20% acetonitrile
- Over approximately 10 minutes
Adjust as necessary.
Basic Compounds
Recommended starting additives:
- 0.1% formic acid
- or
- 0.2% acetic acid
Either isocratic or gradient methods may be used depending on sample complexity.
Troubleshooting Common Problems
Most chromatographic issues are caused by:
- Inadequate equilibration
- Improper mobile-phase composition
- Contaminated solvent lines
- Improper sample solvent
- Injector contamination
- Buffer precipitation
- Failing fittings or tubing
Related Resource
Storage Instructions
When analysis is complete:
- Flush the column with approximately 90:10 organic solvent/water.
- Allow system pressure to return completely to zero.
- Disconnect and cap the column for storage.
Important
Never disconnect a column under pressure. Pressure shock can damage column hardware and negatively affect performance.
Related Resources
Best Practices Summary
- Use HPLC-grade solvents only.
- Degas all solvents.
- Prepare buffers fresh daily.
- Allow adequate equilibration.
- Use filters and guard columns whenever possible.
- Increase water to increase RP retention.
- Increase organic solvent to increase HILIC retention.
- Store in approximately 90:10 organic/water solvent.
- Remove all pressure before column removal.