HPLC Retention time shifting

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

4 posts Page 1 of 1
We have two brand new Waters e2695 HPLCs, they run with the same gradient, solvents, columns, guard columns, etc. One instrument is running fine with no retention time shifts, the other is all over the place.....
-Gradient runs using HPLC grade Water, Acetonitrile and MeOH.
-use a Zorbax C18 column and guard column.
- runs are 40 minutes long
-detector is fixed at 360 nm

The instrument ran for 3 months with no problems. Currently, our retention times have shifted. The beginning compounds shift to the left and the remaining 2/3's of the compounds shift to the left and then to the right. Some have shifted by as much as a minute.

Pressure and delta are normal.

We have changed:

guard column and guard column assembly
check valves - done by waters technician
seal pack - done by waters technician
analytical column
sinker frits for all solvents
in line filter

Any ideas or suggestions are welcome!!!
Turn on a channel for the 2695, this will reveal the pressure over the course of the run. If retention times are shifting around, I can assure you that the pressure will not be as constant as on the other 2695. This means you have a leak or a sticking check valve or some other problem that is just proving to be hard to find.

Is there any salt in your MP? Are you premixing and degassing your MP? Are you running 100% ACN through one of the lines or as a cleanout portion of a gradient?

If you are running lots of ACN, you may benefit from a change to ceramic check valve cartridges. When constantly exposed to nearly pure ACN, the ruby/sapphire ones have problems with sticking and floating.
First thing to try (since you say the other system works perfectly) is to take the column off the known "good" system and use it on the suspect system. What is the result? At least you have narrowed down the problem.

If that does not lead you to the answer, then it is time to assume nothing and use some scientific troubleshooting. I bet this article will help you sort it out.

"HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it" - https://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
Thank you, DR and Multidimensional,
We decided to reboot the instrument and believe it or not, that seems to have worked.....when i suggested it to the analyst, even i didn't think it would do anything, but we have retention times that are spot on....
However, I am apprehensive, so will definitely keep you ideas in mind.
Thanks Again!!
4 posts Page 1 of 1

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