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Ensuring optimum HPLC column performace is important for saving time and cost. The HPLC column peformance and retention characteristics should be monitored at regular intervals using standards. MoniFollowing recommendations are provided for routine HPLC column care and maintainance.
Montoring for following signs of potential breakdown in HPLC column performance.
A. Signs of a Damaged HPLC Column
- Change in column selectivity
- Increased backpressure
- Poor peak shapes such as Peak tailing, splitting, or broadening
- Reduced resolution or retention time shifts
- Loss of efficiency (lower plate number)
If you notice these, try a cleaning or regeneration procedure. If unresolved, the column may need to be replaced.
B. Common Best Practices
- Use High-Purity Solvents
- Always use HPLC-grade solvents and water.
- Filter and degas solvents (use vacuum filtration or inline filters and degassers).
- Maintain Proper pH
- For silica-based columns, keep the pH between 2 and 8 (unless the column is specifically rated for wider pH ranges).
- Extreme pH can damage bonded phases and dissolve silica.
- Protect the Column
- Always use a guard column or inline filter to trap particulates and strongly retained compounds.
- Use sample filtration (0.2 µm or 0.45 µm) to prevent clogging.
C. HPLC Column Usage Tips
- Gradual Solvent Changes
- When switching mobile phases, gradually change solvents to prevent precipitation (e.g., buffer to high organic).
- Flush with intermediate solvents if the solvent polarity is drastically different.
- Flow Rate and Pressure
- Follow the manufacturer’s recommended maximum pressure and flow rate.
- Increase flow rate gradually, especially on startup.
- Temperature Control
- Use a column oven if possible to maintain stable temperatures.
- Avoid sharp temperature fluctuations.
- Avoid Air Bubbles
- Prime the system properly.
- Ensure all solvents are degassed and lines are bubble-free.
D. Cleaning and Regeneration
- Reversed-Phase Columns (e.g., C18)
- Periodic flushing:
- Flush with water → remove salts
- Flush with 100% organic solvent (e.g., ACN or MeOH) → remove hydrophobic contaminants
- Use a regeneration protocol from the manufacturer for heavy contamination
- Periodic flushing:
- Normal-Phase Columns (e.g., Silica)
- Use non-polar organic solvents (hexane, isopropanol).
- Store dry or in the mobile phase (depending on instructions).
Check out detailed column cleaning procedure for additional recommendations.
E. Storage Tips
- Short-Term (Daily/Weekly Use)
- Store in the mobile phase (without buffer) or appropriate storage solvent.
- Avoid storing in aqueous buffers due to microbial growth.
- Long-Term
- Flush out all salts and aqueous components.
- Store in 100% organic solvent (e.g., acetonitrile or methanol).
- Seal column ends tightly with caps.
Note – Always check with your HPLC Column manufacturer for any column specific guidelines.