Troubleshooting Problems with Retention Variation

Possible Cause	Prevention/Solution
Decreasing Retention Times
Loss of bonded stationary phase	Replace column Operate at pH 2-8 for silica-based RP columns
Active groups on a stationary phase	Use organic modifier in the mobile phase Increase buffer strength
Increasing flow rate	Check and adjust the pump flow rate
Column overloaded	Reduce the amount of sample injected Use a column with a larger i.d.
Increasing Retention Times
Changing mobile phase composition	Cover solvent reservoirs Prepare fresh mobile phase
Loss of bonded stationary phase	Replace column
Decreasing flow rate	Check and adjust the pump flow rate Check for leaks in the system, including pump seals
Bubbles in mobile phase	Check flow rate and pressure Degas mobile phase
Fluctuating Retention Times
Insufficient column equilibration	Equilibrate column longer between runs Condition the column with a concentrated sample
Change in mobile phase composition	Check make-up of the mobile phase and make-up new if necessary Check proportioning-valve accuracy
Insufficient buffer capacity	Use buffer concentrations >20mM
Fluctuating column temperature	Stabilize ambient temperature Thermostat the column

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