HPLC Retention Time: 10 Real Reasons Why It Changes
Why Retention Time Changes? 10 Real Lab Reasons Retention time (RT) is one of the most sensitive parameters in HPLC. Even a small change (±0.1–0.5 min) can affect system suitability, assay % and method validity. In this article, I explain 10 real reasons why RT changes and how to fix them — based on QC lab experience. 🔹 1. Mobile phase composition change Even 1–2% shift in organic/water composition changes polarity → RT shifts. Example: Less acetonitrile → peaks become more retained. ✔ Always prepare fresh mobile phase ✔ Use volumetric glassware 🔹 2. pH of the buffer RT depends on ionization of analyte. Small pH drift = big RT change (especially weak acids/bases) ✔ Use calibrated pH meter ✔ Filter + degas buffer ✔ Fresh every 24 hrs 🔹 3. Column aging Column loses efficiency, C18 bonding degrades. Peak shape → tailing RT → slightly increased or unstable ✔ Flush column regularly ✔ Use guard column ✔ Replace after 800–1200 injections (depends) 🔹 4. Temperature variation Retention is t...