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 stationary phase	Use organic modifier in mobile phase Increase buffer strength
Increasing flow rate	Check and adjust pump flow rate
Column overloaded	Reduce amount of sample injected Use column with larger i.d.
Increasing Retention Times
Changing mobile phase composition	Cover solvent reservoirs Prepare fresh mobile phase
Loss of bonded stationary phase	1. Replace column
Decreasing flow rate	Check and adjust pump flow rate Check for leaks in 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 concentrated sample
Change in mobile phase composition	Check make-up of 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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