[Readers Insight] Retention Time Drifts: Why Do They Occur?

In this article, we will discuss several possible causes.

Changes in the mobile phase composition are a common cause of retention time drift. For example, if solvent bottles are not well sealed, the organic component of the mobile phase (e.g., acetonitrile, methanol) will evaporate faster than the aqueous component over time, reducing the organic proportion and changing the mobile phase polarity.

Likewise, changes in mobile phase pH and buffer salt concentration will also affect retention times — for instance, loss of volatile components such as formic acid or ammonia, or absorption of CO₂ from air into alkaline buffer systems, will alter pH. Precipitation of buffer salts can also strongly affect analytes that are sensitive to salt concentration.

Common triggers of mobile phase composition changes in laboratories include:

• When multiple projects or multiple users share an instrument, mobile phase bottles are frequently swapped and sometimes caps are forgotten after exchanging.
• Normal-phase mobile phases are highly volatile and should be tightly sealed with sealing film around the cap during use.
• Visible droplets on the inner wall of the solvent bottle indicate in-bottle evaporation — gently invert or mix the bottle before use.

Retention is closely related to temperature. If the column oven temperature is unstable or a column oven is not used, retention-time drift easily occurs. Additionally, exposure of a column to mobile phases at extreme pH can alter the stationary phase chemistry, causing a severe loss of column efficiency.

In method development, an improperly designed gradient program — specifically failing to allow sufficient time to re-equilibrate the column to the initial gradient conditions — will also produce retention time drift.

During mixing of mobile phases, correct operation of the mixing valves is key to maintaining stable retention times. Malfunctioning or poorly calibrated mixing valves will produce actual mixing ratios that differ from the set values.

Worn pump seals, air bubbles in check valves, or small leaks in the system can reduce the actual mobile phase volume delivered to the column or alter its composition, thereby affecting retention of the target peaks. These instrument-related issues should be checked and corrected before running samples.

Although the injection volume is small (typically ~10–20 μL), changing the sample solvent — even simply switching from methanol to acetonitrile — can still cause retention-time changes. If the sample solvent polarity is lower than the initial mobile phase, solvent effects may occur that change peak shape and retention.

It is recommended to dissolve or make up samples in a solvent with the same composition as the initial mobile phase, or in a solvent of equal or higher polarity.