Common problems and solutions
Common issues, causes, and solutions
| Problems | Probable causes | Solutions |
|---|---|---|
| High column pressure | High flow rate Samples or impurities residue Sample precipitates Column bed collapse Extended column usage Improper equilibration of column Different absorption coefficients of eluent Impure eluent | Reduce the flow rate Clean the packing material Adjust the buffer Repack the column Replace the column or the packing material Increase equilibration time Run a blank gradient Use high-purity HPLC-grade reagents |
| Low resolution | Unsuitable elution conditions, such as steep gradients or high flow rates Improper packing of column Large mixed zone at column head or tail Column overload A portion of the samples or impurities remained unremoved after the cleaning process Larger particle size Poor selectivity Mixed-mode retention caused by the silanol groups on the surface | Change the elution conditions by adopting a slower gradient elution or isocratic elution Check column efficiency and repack the column Increase the height of the packing material in the column or reduce the volume at the end of the column Clean and re-equilibrate the column and reduce the sample loading capacity Perform cleaning operation Replace the packing material with the same type but of smaller particle size Add or regulate ion-pair reagents, or replace with other types of packing material Reduce pH to inhibit silanol or replace the column |
| Samples were eluted before gradient elution | High concentration of the eluent in the initial elution solution Inappropriate pH Sample or impurity residues due to multiple sample loadings | Decrease eluent concentration Adjust pH to increase binding Perform cleaning operation |
| The sample is not eluted during gradient elution process | Low final concentration of eluent Low elution capacity of the eluent pH-induced precipitation The mobile phase is not degassed Bubbles are generated after the mobile phase is mixed Improper packing of column Chromatographic column is not cleaned | Increase the final concentration of the eluent Replace with an eluent that has stronger elution capability Adjust pH to avoid precipitation Fully degas the buffer Isocratic elution is recommended Repack the column Select a strong eluent to clean the chromatographic column |
| Occurrence of ghost peaks | Absorption of the eluent itself Trace amounts of organic impurities are bound to the column, concentrated during equilibration and sample loading, resulting in peaks appearing during elution | Run a blank gradient control, or replace the eluent with one that has no UV absorption Select a strong eluent to wash off the organic impurities bound to the chromatographic column |