HPLC Troubleshooting - Changing Retention Times

If you notice your retention times are variable and changing; common causes are listed below.

Causes	Solutions
Flow rate	- Check the method uses the correct flow rate. Ensure the flow rate is accurate using a flow meter.
Insufficient equilibration	- The reversed phase column should be equilibrated using at least 10 column volumes. If 10 column volumes are insufficient, increase the equilibration time. This should be extended for other techniques such as ion exchange and HILIC.
Poor temperature control	- Check the method uses the correct temperature. Ensure the temperature in the column oven is accurate.
Change in column dimension	Ensure the correct column including dimensions are being used.
Change in column stationary phase environment	- Do not use a column which has ion pairing reagent for other mobile phases due to memory effects. - Stationary phase ‘de-wetted’ (historically incorrectly termed ‘phase collapse’).
Improper mobile phase	- Ensure the mobile phase is accurately prepared. - If using the pump to proportionate the mobile phase, ensure the pump is accurately dispensing mobile phase. - Ensure the correct mobile phase is being used and the correct lines are being chosen on the method.
Instrument leaks	- Check for loose fittings throughout the system.
Air bubble in pump	- Purge pump via purge valve.