Chromatography
Ultra-High Performance Liquid Chromatography (UHPLC)
A high-pressure form of HPLC that uses columns packed with very small particles to give faster runs, sharper peaks and less solvent use. Used in pharma QC for fast assays, impurity profiling and high-throughput testing.
- Pharmaceutical
- Biotech
- Contract research (CRO / CDMO)
- Food & beverage
- Environmental
- Clinical research
TROUBLESHOOTING
Common problems, causes and fixes
Pressure rising steadily over a sequence
Likely causes
- • Particles from samples collecting on the column inlet frit
- • Blocked in-line filter
- • Buffer precipitating in high organic
What to do
- • Replace the in-line filter
- • Filter or centrifuge samples more thoroughly
- • Check buffer solubility at the highest organic content in the gradient
Broad or split peaks on a new column
Likely causes
- • Fitting not fully seated (dead volume)
- • Sample solvent much stronger than the starting mobile phase
- • Injection volume too large for the column
What to do
- • Remake the connection with the correct fitting
- • Dissolve the sample in a solvent close to the starting mobile phase
- • Reduce the injection volume
Peaks look flat-topped or jagged
Likely causes
- • Data rate too low
- • Detector time constant too long
What to do
- • Increase the sampling rate so there are at least 15–20 points per peak
- • Use a shorter filter time constant
Retention times differ from the HPLC method after transfer
Likely causes
- • Different dwell volume
- • Gradient not scaled to the new column
- • Frictional heating in the column
What to do
- • Add an isocratic hold or adjust the gradient start to compensate for dwell volume
- • Re-scale gradient time and flow to the new column dimensions
- • Use a narrower column or control temperature
Carry-over in blanks
Likely causes
- • Weak needle wash
- • Sample adsorbed on the needle seat or rotor seal
What to do
- • Use a stronger or two-solvent needle wash
- • Replace the needle seat or rotor seal
In LimsPulse · Calibration Management
Keep every UHPLC in control: usage logs, calibration and maintenance schedules, and automatic blocking of overdue instruments.