Why Poor Mobile Phase Degassing Causes HPLC Pressure Fluctuations
How to Fix Pressure Ripple, Baseline Noise, and Retention Time Instability
HPLC pressure fluctuations—often observed as pressure ripple, unstable baselines, retention time drift, or intermittent flow—are most frequently caused by poor mobile phase degassing and air entrainment within the solvent delivery system. Dissolved gases expand into bubbles under reduced pressure in pumps and mixers, disrupting flow uniformity and compromising chromatographic reproducibility.
What Are the Symptoms of Poor Mobile Phase Degassing in HPLC?
Common Observable Problems
- Pressure readings that oscillate, ripple, or spike
- Baseline noise or drift at constant flow
- Retention time variability between injections
- Occasional flow interruption or delayed pressure recovery
- Inconsistent gradient performance
Most Likely Root Causes
- Dissolved gases forming bubbles in pump heads or mixers
- Air ingestion due to low solvent level, clogged inlet frits, or non-vented reservoirs
- Degasser malfunction or insufficient vacuum
- Cavitation at the pump inlet
- Worn pump seals or check valves
- Incompatible or aggressive mobile phases affecting wetted components
Why Does Poor Degassing Cause Pressure Ripple and Baseline Noise?
Dissolved gases remain stable at atmospheric pressure but expand rapidly under low pressure or shear inside the HPLC pump and mixing system. These bubbles compress and expand during each pump stroke, leading to:
Flow pulsation
Pressure oscillation
Baseline noise
Gradient composition errors
This mechanism explains why degassing problems often worsen at higher flow rates, lower solvent head height, or during gradient transitions.
How to Plan Mobile Phase Volume to Prevent Air Ingress
Calculate Solvent Volume Before Analysis
Running out of solvent is one of the most common triggers for air entering the system.
Recommended planning approach
Total volume per channel = Flow rate (mL/min) × total run time (min) × average gradient fraction
Add a 1.5–2× safety factor for priming, purging, equilibration, and repeat injections
Best practices
- Prepare all mobile phases at the same time
- Use reservoirs large enough to maintain solvent well above inlet frits
- Never allow a solvent line to run dry during operation
If a pump runs dry
- Stop the run immediately
- Purge all solvent lines and pump heads with fresh mobile phase
- Resume only after pressure and baseline stability are restored
Best Practices for Degassing Mobile Phases in HPLC
Accepted Degassing Methods
In-Line Vacuum Membrane Degasser
- Confirm the degasser is enabled and pulling stable vacuum
- Monitor instrument diagnostics for vacuum warnings or instability
- Reduced vacuum performance correlates directly with pressure ripple
Helium Sparging
- Use low, controlled helium flow through a frit before connection
- Avoid continuous high-rate sparging during analysis to minimize evaporation and pH drift
Vacuum Degassing and Sonication
- Primarily applicable to aqueous buffers
- Sonicate briefly under vacuum
- Always filter after degassing
Degassing Workflow Rules
- Degas aqueous and organic components separately
- Degas the final mixture if prepared off-line
- Filter through 0.2–0.45 µm membranes after degassing
- Ensure all reservoir caps are properly vented
- Avoid surfactants, which impair degasser membrane function
How to Fix HPLC Pressure Fluctuations After Air Enters the System
Step-by-Step Recovery Procedure
01
Stop the run and reduce the flow rate
02
Open the purge valve
03
Prime each solvent channel individually
- Inspect inlet tubing and frits
- Replace clogged frits if flow is restricted
- Purge until flow is continuous and bubble-free
04
Prime pump heads according to system protocol
- For dual-head pumps, prime each head independently
05
Continue purging until pressure stabilizes without oscillation
06
Re-equilibrate with fresh mobile phase
- Flush 10–20 column volumes or per column guidance
How to Diagnose Degasser vs Pump Problems in HPLC
Interpret the Pressure Pattern
High-frequency ripple
air in pump heads or pulsation
Irregular spikes or drops
intermittent air ingress or inlet restriction
Composition-linked changes
mixing or compressibility issues
Isolation and Diagnostic Checks
- Bypass the column Replace with a short capillary or backpressure restrictor (~20–50 bar)
- Persistent fluctuation indicates pump, degasser, or inlet origin
- Change flow rate Increasing ripple at higher flow suggests cavitation or degasser issues
- Inspect solvent inlet path Replace frits, check tubing alignment, confirm venting
- Evaluate degasser condition Flush channels with water followed by methanol or isopropanol if permitted
- Inspect pump check valves Sticky or worn valves cause erratic pressure behavior
- Inspect seals and plungers Look for leaks, salt residue, or solvent accumulation
- Confirm mixing system function
- Low-pressure mixing: valve timing and trapped air
- High-pressure mixing: priming balance and compressibility settings
- Verify pulse dampers if installed
Mobile Phase Preparation Factors That Affect Pressure Stability
Buffer Preparation
- Fully dissolve salts before final volume adjustment
- Allow solutions to reach room temperature
- Confirm solubility at intended organic content
- Prevent precipitates that clog inlet frits
Organic Modifiers
Acetonitrile: lower viscosity but still requires thorough degassing
Methanol: higher viscosity and more prone to cavitation if poorly degassed
pH Compatibility
- Use mobile phase pH ranges compatible with instrument and column limits
- Avoid prolonged exposure to extreme pH unless explicitly supported
Preventive Practices to Avoid Future Pressure Fluctuations
Maintain adequate solvent head height at all times
Replace inlet frits routinely
Clean proportioning valves on a schedule
Monitor baseline noise trends as early warning indicators
Purge thoroughly after every mobile phase change
Define acceptance criteria (e.g., pressure stability within ±2–5 bar)
Document retention time and gradient reproducibility
Verification After Corrective Actions
With the column bypassed, confirm stable pressure at fixed flow
Reinstall the column and verify nominal backpressure
Run short test injections to confirm baseline and retention stability
Safety and Compatibility Considerations
Extended exposure to strong acids or bases can damage pump seals, plungers, and valve materials. Always confirm mobile phase compatibility with both the HPLC system and column before use.
Frequently Asked Questions (FAQ)
Can poor degassing damage an HPLC pump?
Yes. Repeated cavitation and air compression can accelerate wear of seals, check valves, and plungers.
Does methanol require more degassing than acetonitrile?
Methanol's higher viscosity increases cavitation risk, making thorough degassing especially important.
How do I know if my degasser is failing?
Persistent pressure ripple, increased baseline noise, or unstable degasser vacuum readings indicate reduced performance.
Should I degas organic solvents even if they look clear?
Yes. Visual clarity does not indicate absence of dissolved gases.
Final Summary
Poor mobile phase degassing is one of the most common and preventable causes of HPLC pressure instability.
Adequate solvent planning, proper degassing, vented reservoirs, thorough priming, and structured diagnostics reliably restore stable pressure, low baseline noise, and reproducible chromatography.