Even the best HPLC methods sometimes run into trouble.
Poor peak shapes? pressure spikes? incorrect retention times?
You need to identify define the problem, define which component(s) may be causing it, then solve it. This process is called troubleshooting.
It’s an art as much as it is a science.
Think of troubleshooting like being a scientific detective: observe, hypothesize, and test.
Here’s a simple way to start:
1. Start by Observing: What’s Wrong?
Baseline noise?
Shifting retention times?
High or unstable pressure?
Peak splitting, tailing, or missing peaks?
The symptoms are your first clues.
2. Group the Possible Causes:
Pressure Issues:
- Blocked component, filters, or tubing.
- Precipitated sample or buffer salts.
- Column blockage or collapse.
- Pump problems (e.g., seal, head)
- Leaks.
- Air trapped in the system.
Peak Problems (Tailing, Broadening, Splitting):
- Damaged or incorrect column chemistry.
- Injection solvent mismatch.
- Sample solubility issues.
- Mobile phase problems (pH, expired solvents, composition, flow rate).
- Contamination.
- Incorrect detector settings.
Retention Time Shifts:
- Mobile phase composition changes.
- Column degradation or overload.
- Temperature fluctuations.
- Air trapped in the pump or system
- Injection solvent mismatch.
Baseline Problems (Noise, Drift):
- Detector issues (e.g., dirty flow cell, lamp aging).
- Air bubbles in the system.
- Mobile phase problems.( contamination, non- homogeneous, improper mixing, …)
- Slow or incomplete column equilibration
- Strongly retained sample components.
- Leaks
- Pump pulsation
- Column leaking silica or packing materials
3. Fix Systematically:
- Check simplest things first (mobile phase freshness, leaks, air bubbles).
- Flush the system, sometimes a cleaning run saves hours!
- Review method conditions a small parameter shift can cause problems.
- Test with a known good column to isolate the issue.
- Replace consumables (filters, seals) if needed
Pro Tip:
- Never change too many variables at once.
- Change one thing, observe, then move to the next. Good troubleshooting is patient and methodical.
HPLC is an incredibly powerful tool, but it relies on precision and care.
When things go wrong, and they will, stay calm, think like a scientist, and let the data guide you to the answer.
What was the trickiest HPLC issue you’ve ever had to troubleshoot? I’d love to hear your stories in the comments!
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Resource Person: Abanoub Efraim