Retention time shifts

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Probable cause		Suggested actions
1	NOTE: Use ten column volumes of equilibration during method development. For established methods, the equilibration time can be reduced with optimization.	Determine the column void volume and system dwell volume to determine the optimal equilibration time for the gradient method.
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.	Check the number of injections does not exceed the typical number of injections for this column and application. Replace the column 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: Use high purity HPLC grade or better solvents only. Solvents from different suppliers might have subtle differences in their contamination profile.	Switch to a fresh batch of solvents.
5	Column/mobile phase incompatibility with the analyte	Change the chemistry of the column to strengthen interactions with the analyte. Choose the pH of the eluent that is not too close to the pKa value of the analyte. Ensure that the pH of the eluent within the column manufacturer’s specifications.
6	Insufficient flushing after use of buffer	Extensively flush the system with water prior to switching from methods using buffer to methods using organic solvents.

21-Jun-2025