Decreasing retention

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Probable cause		Suggested actions
1	NOTE: The eluent pH and the column temperature shall never exceed the manufacturer‘s specifications. Use specialty columns when working at high temperatures and/or pH extremes. NOTE: Most silica-based columns degrade rapidly at pH below 2 or above 8, especially at temperatures higher than 40 °C.	Check the number of injections does not exceed the typical number of injections for this column and application. Replace the column if necessary.
2	NOTE: The eluent pH and the column temperature shall never exceed the manufacturer‘s specifications. Use specialty columns when working at high temperatures and/or pH extremes. NOTE: Most silica-based columns degrade rapidly at pH below 2 or above 8, especially at temperatures higher than 40 °C.	Verify that the column chemistry is suitable for the given analytical problem. Switch to another column type if necessary.
3	NOTE: The eluents should be miscible and should not form salt precipitates over the range of the entire gradient. The eluents should also be properly degassed. NOTE: Due to its miscibility with water and most organic solvents, isopropanol is often the choice when removing air bubbles from an HPLC system.	Ensure that the method settings are properly set and the gradient system is delivering the correct gradient composition. Check if the mobile phase reservoirs are empty or if the filter frits are blocked. Check for air bubbles in the system. If necessary, purge the pump and system with a viscous mobile phase to remove any air bubbles. Check the system for any leaks. Check for leaking pump seals. If necessary, replace pump seals. Check for sticking check valves. If necessary, sonicate or replace check valves.
4	NOTE: Using a guard column and an inline filter protects the separation column from premature failure. NOTE: Using a suitable inline filter (e.g. 0.2 or 0.5 µm) and always filtering samples and eluents prevent particulate material from entering your HPLC system.	Flush and, if necessary and allowed by the supplier, backflush the column. Replace the column if necessary. If using a guard column or inline filter, replace it.
5	Column overload	Reduce the injection volume or dilute the sample. Switch to a column with a higher sample capacity (increase length or diameter).
6	Improper method settings	Check temperature settings in the column compartment.

21-Jun-2025