Problem |
Possible cause |
Solution |
Broad peaks |
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 in. i.d.); connect tubing with matched fittings |
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 |
Contamination |
Flush column to remove contaminatint; use HPLC-grade solven |
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. |
Negative peaks |
Refractive index detection - refractive index of solute less than that of mobile phase |
Reverse polarity to make peak positive |
UV-absorbance detection - absorbance of solute less than that of mobile phase |
Use mobile phase with lower UV absorbance; if recycling solvent, stop recycling when recycled solvent affects detection |
Peak Doubling |
Blocked Frit |
Replace or clean frit; install 0.5-um porosity in-line filter between pump and injector to eliminate mobile-phase contaminants or between injector and column to eliminate sample contaminants |
Coelution of interfering compound |
Use sample cleanup or prefractionation; adjust selectivity by changing mobile or stationary phase |
Coelution of interfering compound from previous injection |
Flush column with strong solvent at end of ran; end gradient at higher solvent concentration |
Column overloaded |
Use higher-capacity stationary phase; increase column diameter; decrease sample amount |
Column void or channeling |
Replace column, or, if possible, open top endfitting and clean and fill void with glass beads or same column packing; repack column |
Injection solvent too strong |
Use weaker injection solvent or stronger mobile phase |
Sample volume too large |
Use injection volume equal to one-sixth of column volume when sample prepared in mobile phase for injection |
Unswept injector flow path |
Replace injector rotor |
Peak Fronting |
Channeling in column |
Replace or repack column |
Column overloaded |
Use higher-capacity stationary phase; increase column diameter; decrease sample amount |
Tailing Peaks |
Basic solutes - silanol interactions |
Use competing base such as triethylamine; use a stronger mobile phase; use base-deactivated silica-based reversed-phase column; use polymeric column |
Beginning of peak doubling |
See peak doubling |
Chelating solutes - trace metals in base silica |
Use high purity silica-based column with low trace-metal content; add EDTA or chelating compound to mobile phase; use polymeric column |
Silica-based column - degradation at high pH |
Use polymeric, sterically protected, or high-coverage reversed-phase column; install silica gel saturatorcolumn between pump and injector |
Silica-based column - degradation at high temperature |
Reduce temperature to less than 50 C |
Silica-based column - silanol interactions |
Decrease mobile-phase pH to suppress silanol ionization; increase buffer concentration; derivatize solute to change polar interactions |
Unswept dead volume |
Minimize number of connections; ensure injector rotor seal is tight; ensure all compression fittings arecorrectly seated |
Void formation at head of column |
Replace column, or, if possible, open top endfitting and clean and fill in void with glass beads or samecolumn packing; rotate injection valve quickly; use injection valve with pressure bypass; avoid pressure shock |
Spikes |
Bubbles in mobile phase |
Degas mobile phase; use back-pressure restrictor at detector outlet; ensure that all fittings are tight |
Column stored without caps |
Store column tightly capped; flush reversed-phase columns with degassed methanol |