Help on: Retention Time Shifts in NP-HPLC

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Question:
“I am using NP-HPLC, my analyte needs 100% hexane. I am using pure material with un-known RT. The RT shifts to the right with every injection, something like 2-2.5 min for each injection. I started at 9 min and get to 16 min in 6 injections. Then I decided to clean the column overnight with 100% hexane and now I started from 27 min and it's shifting ... Any advice will be helpful. The temperature is constant, there is only one peak for my analyte.”

Answer:
“The most common problem when using NP-HPLC is retention time variability. This is normally due to the presence of very polar molecules such as water in the mobile phase. A small amount of water can significantly alter retention times. Once your column has equilibrated with the water content in your mobile phase any alteration in this amount will cause shifts in retention time. Reaching full equilibration is very time consuming in normal phase systems and can take anything from a few hours to a few days.

It sounds like your original injections had drifting retention times because the column had not reached equilibration. A strategy of running the mobile phase through the column overnight should help to achieve equilibration but since the RTs have not settled down either the column has not yet equilibrated or perhaps the mobile phase was altered. Is the mobile phase 100% hexane?

Controlling the water content of the mobile phase is desirable for reproducible results. Working with dry solvents is usually difficult and results in very long equilibration times. Using solvents which are fully saturated with water causes water to build up in the pores of the column. A common approach is to use ‘half-saturated’ solvents. Add 1 or 2 mL of water to 500mL of dry solvent and stir for 30 minutes, then remove excess water and combine with 500mL of dry solvent. Use this as your mobile phase and your equilibration should be quicker (although it may still take a few hours). If you can dedicate the column to your method then equilibration should be quicker when you use the column again in the future.

A possible solution for shifting retention times and equilibration problems that I have heard works well (but have not personally tried) is to use a magnetic stirrer to stir the mobile phase gently throughout the run, making sure not to place it close to the detector since it may cause baseline ripples.

Another potential source of water is your sample, if you suspect it may contain water then it is a good idea to dry it with anhydrous sodium sulfate of a high purity grade.”