Overview
Microbial contamination can develop in liquid chromatography systems whenever predominantly aqueous mobile phases are used, particularly under ambient light and room-temperature conditions. Microorganisms can colonize solvent reservoirs, tubing, pumps, mixers, degassers, autosamplers, and columns. Cells and biofilms shed both particulate matter and soluble metabolites that enter the flow path. These materials are frequently smaller than typical column inlet frit pore sizes and can pass through, accumulate within the packed bed, and alter chromatographic performance.
Once microbial growth is established, contamination often becomes system-wide, making remediation difficult and time-consuming. The resulting effects include ghost peaks, increased backpressure, retention-time instability, poor reproducibility, and compromised pump and valve performance. For this reason, prevention is considerably more effective than corrective action.
Root Causes and Risk Factors
Use of mobile phases containing very high water content, particularly without organic modifiers
Exposure of clear solvent reservoirs to ambient or laboratory lighting
Extended idle periods with water-filled reservoirs and solvent lines
Poor reservoir hygiene, including topping off old solutions or reusing aged mobile phases
Insufficient filtration of aqueous solvents
Predominantly aqueous autosampler wash solutions
Introduction of microbial inoculum through samples or sample containers
Corrective Actions: System Decontamination
Important: Perform all cleaning with the analytical column removed and a bypass union installed. Observe manufacturer limits for solvents, flow rates, and materials compatibility.
A. Flow Path Flushing
- Discard contaminated mobile phases and replace with freshly prepared, filtered solvents
- Flush degasser and pump sequentially at moderate flow:
- High-purity water to dissolve soluble residues
- Organic-rich solvent to disrupt biofilms and remove hydrophobic material
- Water to remove residual organic solvent
- Cycle mixer and proportioning valve compositions to flush all channels
- Flush autosampler injection valve, sample loop, and wash lines with organic-rich solvent
B. Component-Level Maintenance
- Clean solvent reservoirs and caps using laboratory detergent followed by thorough rinsing
- Replace or clean solvent pickup frits showing discoloration or fouling
- Install or replace inline filters to capture particulates
- Clean or service check valves if pressure ripple persists
- Flush degasser channels with compatible solvents within manufacturer specifications
C. Column and Guard Handling
- Replace guard columns if backpressure increase or ghost peaks persist
- For the analytical column:
- Flush forward with strong organic solvent at reduced flow
- Add a small fraction of isopropanol if compatible to disrupt biofilms
- Avoid backflushing unless explicitly permitted by the manufacturer
- Re-equilibrate thoroughly and reassess performance
Verification After Remediation
Run solvent-only blanks to confirm baseline cleanliness
Inject mobile-phase blanks to verify absence of ghost peaks
Perform gradient tests to confirm stable backpressure and retention
Record baseline noise, pressure, and retention metrics for future comparison
Common Pitfalls and Cautions
Biocides may damage system components or interfere with detection and should not be used unless explicitly approved
Chlorine-containing cleaners and strong oxidizers are incompatible with stainless-steel flow paths
Unauthorized backflushing can permanently damage columns
Inline filters reduce risk but do not fully prevent microbial deposition within the column bed
Summary
Prevention is Key
Microbial contamination in aqueous mobile phases can severely compromise chromatographic performance and system reliability. The problem often propagates throughout the instrument and is difficult to eliminate once established.
Effective diagnosis relies on blank testing, visual inspection, column bypass experiments, and assessment of pump behavior. Remediation requires staged flushing with organic-rich solvents, replacement of sacrificial components, and careful column handling. Long-term control depends on modifying mobile phase composition, protecting solvents from light, maintaining reservoir hygiene, and avoiding prolonged idle periods with water-filled systems.
