Possible Cause Solution
| Buffer retention times | Use buffer with concentration greater than 20 mM. |
| Contamination buildup | Flush column occasionally with strong solvent |
| Equilibration time insufficient for gradient run or changes in isocratic mobile phase | Pass at least 10 column volumes through the column for gradient regeneration or after solvent changes |
| First few injections - active sites | Condition column by injecting concentrated sample |
| Inconsistent on-line mobile-phase mixing | Ensure gradient system is delivering a constant composition; compare with manually prepared mobile phase; partially premix mobile phase |
| Selective evaporation of mobile-phase component | Cover solvent reservoirs; use less-vigorous helium purging; prepare fresh mobile phase |
| Varying column temperature | Thermostat or insulate column; ensure laboratory temperature is constant. |