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Ten Common Chromatography Problems · Causes and Fixes
Possible cause
- A dry or voided bed head (most common)
- A blocked frit causing uneven flow distribution
- Overloading (beyond column capacity)
- Injection solvent mismatched to the mobile phase
- Excessive extra-column dead volume (fitting problem)
Fix
- Replace the guard cartridge and see whether it improves
- Regenerate by flushing with a strong solvent (isopropanol) to clear the blockage
- Reduce the load (usually < 10 μg on an analytical column)
- Switch to a weaker injection solvent (the initial mobile phase)
- Check every fitting and minimize dead volume
Possible cause
- Basic compounds interacting with residual silanols
- Mobile phase pH not suppressing ionization of the basic compound
- A contaminated or damaged bed head
- Injection solvent too strong
Fix
- Add 0.1% triethylamine (TEA) to the mobile phase to mask silanols
- Lower the mobile phase pH (pH < pKa – 2) to suppress protonation of the basic group
- Switch to a more completely endcapped column (HPLCONE is fully endcapped)
- Dissolve the sample in a weaker solvent
Possible cause
- A void at the bed head (bed collapse)
- Injection solvent far stronger than the mobile phase
- Cracking within the packed bed
Fix
- Inspect the bed head: remove the end fitting and look for a void; a void means the bed head is damaged and the column must be replaced
- Dissolve the sample in a weaker solvent (similar to or weaker than the initial mobile phase)
- If the bed has cracked, the column must be replaced
Possible cause
- The inlet frit blocked by particulates
- Buffer salt crystallizing out
- Protein or lipid deposited at the bed head
- Mobile phase too viscous (neat methanol at low temperature)
- Blocked tubing (not the column itself)
Fix
- Remove the column first and see whether system backpressure drops; if it does, the problem is in the column
- Flush with isopropanol at high flow for 20 min
- Replace the guard cartridge or the inlet frit
- Review sample preparation and ensure 0.22 μm filtration
- Raise the temperature (40 °C) to reduce methanol viscosity
Possible cause
- A loose or cracked fitting leaking
- A pump problem (failed check valve)
- Loss of packing from the column (broken frit)
Fix
- Check every fitting for leakage
- Check that pump pressure is stable (replace the check valve ball)
- If packing has been lost, check whether the outlet frit is intact
Possible cause
- Bubbles in the mobile phase
- An aged detector lamp (deuterium lamp at end of life)
- Mobile phase not adequately degassed
- Changing organic proportion during gradient elution
- Column not adequately equilibrated
Fix
- Degas the mobile phase by sonication for 15 min, or use in-line degassing
- Check the deuterium lamp energy and replace it when below threshold (our compatible lamps can be used)
- For gradient methods, run a blank gradient first and record the baseline background
- Extend equilibration to 20–30 column volumes
Possible cause
- Carryover from the previous injection (cross-contamination)
- Impurities in the sample solvent
- Trace UV-absorbing impurities in the mobile phase
- A contaminated injection valve
Fix
- Extend the wash time, or flush the injection valve with a strong solvent
- Check that the sample solvent blank is clean
- Switch to HPLC-grade reagents for the mobile phase
- Flush the injection valve with isopropanol
Possible cause
- Unstable column temperature
- Inconsistent mobile phase composition
- Column not adequately equilibrated
- Unstable pump flow rate
Fix
- Use a column oven and fix the temperature
- Prepare the mobile phase accurately, making up to volume in a volumetric flask
- Equilibrate with at least 10 column volumes before each injection
- Calibrate the pump flow rate
Possible cause
- Inaccurate injection volume (autosampler problem)
- Insufficient detector lamp energy
- Sample lost in the autosampler (adsorption)
- Sample concentration miscalculated
Fix
- Verify the injection volume with a standard of known concentration
- Check and replace the detector deuterium lamp
- Switch to PEEK sample tubing to reduce adsorption
- Prepare a fresh standard to verify the concentration
Possible cause
- Sample solvent absorbs less than the mobile phase (negative peak)
- Refractive index mismatch (RI detector)
- Overloading producing a system peak
Fix
- Dissolve the sample in a solvent close to the mobile phase
- Reduce the injection volume
- On an RI detector, refill the reference cell with the same mobile phase as the sample side