What are common causes of LC column plugging, fouling and/or high pressure problems? - WKB121927

ENVIRONMENT

• Column plugging
• High back pressure
• Troubleshooting

ANSWER

Common causes of column plugging, which lead to premature high pressure problems, include the following:

1. Microbial growth/contamination (most common problem)

2. Nonbiological particulates in mobile phase

3. Nonbiological particulates shedding from wearable system parts (like the pump seals)

4. Sample and/or sample matrix building on column (requiring column cleaning)

5. Particulates in samples being injected

6. Precipitation of analytes, if not soluble in mobile phase

7. Precipitation of buffer salts if exposed to high organic too quickly

8. Flow rate is too fast (for example, if column is 1.0 mm I.D., a flow rate of 0.6 mL/min will cause the column to overpressure).

9. Crimped/crushed reusable fitting piece (like Peek ferrule or on-piece Finger tight fitting)

ADDITIONAL INFORMATION

•  If the issue is microbial contamination:
  • The column cannot be cleaned properly, since microbes have now embedded themselves into the packed bed. 
    • Even reverse-flushing will not clean the column if it has been contaminated by microbes. 
      • To correct this problem, discard the solvent bottle filters (sinkers), decontaminate your LC system (according to the manufacturer's guidelines), and install new sinkers.  
        • Proper mobile phase handling is required in order to lower the risk of microbial contamination, including:
          • Replace low salt molarity aqueous mobile phases (such as 10mM) every 24-48 hours unless it has at least 10% organic in the bottle. If it contains a minimum of 10% organic, it can be replaced every week provided the organic has not evaporated extensively.
          • For Size Exclusion Chromatography (SEC methods) using Waters BEH SEC columns, refer to the Mobile Phase handling section in the Guide to Size-Exclusion Chromatography (SEC) of mAb Aggregates, Monomers, and Fragments
            • See related video: Do you have to change aqueous mobile phase every day? | Trust your Science 7 - Highlights how microbial growth can contaminate system in ~2 days and disrupt chromatography.
            • See related video: Attack of the column killing aqueous mobile phase bacteria! | Trust Your Science 18 - Highlights how a green laser can help you “see” bacterial growth in mobile phase.  Also shows impact of microbial contamination on SEC columns.
• If nonbiological particulates (from mobile phase, instrument, sample, etc..) have plugged the column:
  • You might be able to fix the issue by reverse-flushing your column at 0.1mL/min into a beaker (not into your detector). 
    • To prevent nonbiological samples from plugging a column:
      • Filter mobile phases with 0.2µm filtration for <3.5µm particle size columns.
      • Filter mobile phases with 0.45µm filtration for 3.5µm particle size columns and higher.    
      • Use of a pre-column inline filter or guard cartridge can also help prevent a column from plugging from nonbiological particulates.
      • Determine if your instrument Preventive Maintenance (PM) was performed within the past 12 months.
        • Pump seals need to be replaced every year (sometimes more often) because they wear over time and can contribute to microparticulates being introduced into the mobile phase and then onto the inlet frit of your column.
• If sample matrix has built up on the column:
  • The column might be able to be cleaned with appropriate solvents as per the column care and use manual.
• If something has precipitated on the column:
  • You may be able to run a very long, shallow gradient, in order to resolubilize the precipitate.