Troubleshooting Problems with Retention Variation

Possible Cause


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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