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Why Must the Mobile Phase Be Filtered and Degassed?
Release time:
2025-06-20
I. Why Filtration is Essential?
Particulates in mobile phases cause:
Column Clogging:
Particles accumulate on inlet frits (0.5-2μm pores), increasing backpressure (>500 psi)
Reduced efficiency: Plate count drops >20% with peak broadening
System Damage:
Pump seal wear: Particles accelerate piston seal degradation
Injector jamming: >10μm particles scratch rotor seals
Baseline Noise: Light scattering in UV flow cells
▌ Filtration Protocol:
Membrane Selection:
| Solvent Type | Recommended Membrane | Pore Size |
|---|---|---|
| Aqueous/Buffers | Nylon 66 | 0.45μm |
| Organics (ACN/MeOH) | PTFE | 0.22μm |
Apparatus:
Use glass filtration units (organic resistance) at ≤0.3MPa vacuum
Critical Notes:
Filter buffers AFTER salt dissolution (Never add salts post-filtration!)
Change membranes when switching solvents
II. Science Behind Degassing
Dissolved gases (O₂, N₂, CO₂) induce:
Pump Failures:
Bubbles cause pressure pulsation (±10% fluctuation)
Accelerated plunger wear in low-pressure mixing systems
Detector Interference:
UV baseline spikes: Refractive index changes from bubbles
Signal drift in electrochemical detection (O₂ redox reactions)
Retention Time Shifts:
Bubbles occupy ~5% column volume, altering flow paths
▌ Degassing Methods Comparison:
| Method | Efficiency | Application | Limitations |
|---|---|---|---|
| Sonication | 60-70% | Small volumes (<2L) | 40% regas in 30 min |
| Helium Sparging | >95% | Oxygen-sensitive analyses | High helium cost |
| Online Degasser | 98% | UHPLC/Gradient analysis | Membrane replacement |
| Vacuum Filtration | 80% | Buffer solutions | Volatile loss |
▶ Best Practices:
Staged Degassing:
Step 1: Sonicate 20 min (40kHz, 25°C)
Step 2: Helium sparge 15 min (0.5L/min)
Step 3: Vacuum filter through 0.22μm membrane
Online Degasser Maintenance:
Weekly flush with 0.1M HNO₃ (30 min)
Replace PTFE tubing every 6 months
III. Troubleshooting Degassing-Related Failures
Case 1: Periodic Baseline Fluctuation (>0.5mAU)
Diagnosis: Microbubbles in pump head causing flow instability
Solution:
Purge pump at 5mL/min for 10 min with purge valve open
Increase degasser temp to 35°C (for ACN/water)
Case 2: Retention Time Drift (>±0.2min)
Root Cause: CO₂ dissolution forming carbonic acid (pH shift)
Corrective Actions:
Prepare buffers fresh, store under nitrogen
Add 0.001% triethylamine (suppresses CO₂ absorption)
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