Change or variation in Retention time could be due to changes in the Mobile phase composition, column chemistry, column temperature or flow rate. Error in on-line mixing of isocratic or gradient mobile phase can also cause retention time issues.
🌺 Mobile phase composition
Mobile phase composition changes usually occur when the original mobile phase composition is not followed while preparation and mixing of buffer-organic solvents in its actual proportion. In rare cases, evaporation of one mobile phase component may occur.
🏵 Column chemistry
Column chemistry changes will occur over the lifetime of the column and generally are gradual over several weeks or months. Column ageing usually is accompanied by rising column backpressure, gradually shifting retention times (longer or shorter) and more peak tailing. Exchange the column for a new one to confirm a column-ageing problem.
🌹 Column temperature
Column temperature changes can cause retention time changes of 1-3% per 1°C change in temperature. When a column oven is not used (i.e. “ambient” conditions), the temperature often cycles over the course of the day (and night) due to laboratory temperature changes. Although the laboratory temperature may seem to be constant, as measured at the room’s thermostat, the micro-environment at the HPLC system may change significantly, especially if a heating or air conditioning vent blows directly at the system.
☘ Flow rate
Flow rate problems can be due to bubbles, leaks or pump problems. Bubble problems will correlate with low or fluctuating pressure and increased retention times. Degas the mobile phase, then purge the pump by opening the purge valve and pumping of mobile phase through the pump at several times the normal flow rate to displace bubbles.
Change or variation in Retention time could be due to changes in the Mobile phase composition, column chemistry, column temperature or flow rate. Error in on-line mixing of isocratic or gradient mobile phase can also cause retention time issues.
🌺 Mobile phase composition
Mobile phase composition changes usually occur when the original mobile phase composition is not followed while preparation and mixing of buffer-organic solvents in its actual proportion. In rare cases, evaporation of one mobile phase component may occur.
🏵 Column chemistry
Column chemistry changes will occur over the lifetime of the column and generally are gradual over several weeks or months. Column ageing usually is accompanied by rising column backpressure, gradually shifting retention times (longer or shorter) and more peak tailing. Exchange the column for a new one to confirm a column-ageing problem.
🌹 Column temperature
Column temperature changes can cause retention time changes of 1-3% per 1°C change in temperature. When a column oven is not used (i.e. “ambient” conditions), the temperature often cycles over the course of the day (and night) due to laboratory temperature changes. Although the laboratory temperature may seem to be constant, as measured at the room’s thermostat, the micro-environment at the HPLC system may change significantly, especially if a heating or air conditioning vent blows directly at the system.
☘ Flow rate
Flow rate problems can be due to bubbles, leaks or pump problems. Bubble problems will correlate with low or fluctuating pressure and increased retention times. Degas the mobile phase, then purge the pump by opening the purge valve and pumping of mobile phase through the pump at several times the normal flow rate to displace bubbles.