Troubleshooting High Performance Liquid Chromatography
Do you have a problem with any of the following topics? 1.Peaks 
2.Leaks
3.Recovery
4. Sensitivity
5. Retention
Changing retention times
Decreasing retention times
Increasing retention times
Retention beyond total permeation volume
6. Equilibration
7.Baseline
8. Pressure
Broad Peak Causes and Solutions
Cause Solution
| Analytes eluted early due to sample overload | Dilute sample 1:10 and reinject |
| Detector-cell volume too large | Use smallest possible cell volume consistent with sensitivity needs; use detector with no heat exchanger in system |
| Injection volume too large | Decrease solvent strength of injection solvent to focus solute; inject smaller volume |
| Large extra column volume | Use low- or zero-dead-volume endfittings and connectors; use smallest possible diameter of connecting tubing (<0.10> |
| Mobile-phase solvent viscosity too high | Increase column temperature; change to lower viscosity solvent |
| Peak dispersion in injector valve | Decrease injector sample loop size; introduce air bubble in front and back of sample in loop |
| Poor column efficiency | Use smaller-particle-diameter packing, lower-viscosity mobile phase, higher column temperature, or lower flow rate |
| Retention time too long | Use gradient elution or stronger isocratic mobile phase |
| Sampling rate of data system too low | Increase sampling frequency |
| Slow detector time constant | Adjust time constant to match peak width |
| Some peaks broad - late elution of analytes retained from previous injection | Flush column with strong solvent at end of run; end gradient at higher solvent concentration |
Ghost Peaks: Causes and Solution
Possible Cause Solution
| Contamination | Flush column to remove contaminatint; use HPLC-grade solvent |
| Elution of analytes retained from previous injection | Flush column with strong solvent at end of run; end gradient at higher solvent concentration. |
| Ion-pair chromatography - upset equilibrium | Prepare sample in mobile phase; reduce injection volume |
| Oxidation of trifluoroacetic acid in peptide mapping | Prepare trifluoroacetic acid solutions fresh daily; use antioxidant |
| Reversed-phase chromatography - contaminated water | Check suitability of water by running different amounts through column and measure peak height of interferences as function of enrichment time; clean water by running it through old reversed-phase column; use HPLC-grade water. |
| Unknown interferences in sample | Use sample cleanup or prefractionation before injection. |
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