What are common causes of low resolution for a liquid chromatography (LC) column? - WKB244143
Article number: 244143
ENVIRONMENT
- Column performance
- Low resolution
- Wide peaks
- Matrix
ANSWER
Common causes of low resolution include (but are not limited to):
- System dispersion (band spread, band broadening) is too high.
- Too much system volume between the injector and the detector leads to wider peaks and, sometimes, tailing.
- New flow cell that has more volume than the old flow cell
- New tubing that has more volume than the old tubing
- Too much system volume between the injector and the detector leads to wider peaks and, sometimes, tailing.
- Poor connections in the flow path between the injector and the detector:
- Incorrect (too short) ferrule depth can cause voids inside the end fitting.
- Make a new connection.
- Slipped ferrule (tubing can slip inside the end fitting, causing a void)
- Can occur when using PEEK finger-tight fittings.
- Install a new fitting.
- Incorrect (too short) ferrule depth can cause voids inside the end fitting.
- Non-specific binding:
- Proteins and acidic analytes can adsorb to stainless steel surfaces (through cationic attraction) and to the particles, as well through hydrophobic or hydrophilic attraction (depending on particle type):
- Make several "conditioning injections" with a high concentration of the analyte to saturate "active sites" on the stainless steel and particle surfaces to improve recovery and resolution.
- Proteins and acidic analytes can adsorb to stainless steel surfaces (through cationic attraction) and to the particles, as well through hydrophobic or hydrophilic attraction (depending on particle type):
- Matrix component buildup on the column:
- Sample matrix may foul columns, building up on the column injection to injection.
- Perform column cleaning procedure.
- Sample matrix may foul columns, building up on the column injection to injection.
- Microbial growth/contamination:
- May cause peak tailing but more often causes split peaks, lower resolution, and high-pressure problems
- Replace high-aqueous mobile phases every 24-48 hours if possible when running 2.5-µm and sub-2µm particle columns.
- May cause peak tailing but more often causes split peaks, lower resolution, and high-pressure problems
- System problems (including but not limited to):
- Incorrect flow rate being delivered by the system
- Incorrect column temperature
- Incorrect solvent mix
ADDITIONAL INFORMATION
Waters now offers MaxPeak Premier columns, which prevent acidic analytes from adsorbing to the stainless steel surfaces inside the columns.
See: MaxPeak Premier Columns Web Page
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