I agree with John. Notice it is the late eluters that shift the most but the profile is the same. You may have to examine your strongest most organic solvent (Mobile B) and increase it pH buffering capacity.

"It's also curious that not much elutes until 5 minutes":

To address such a statement requires knowledge of the method used. NO HPLC method conditions are reported. Any constructive discussion which might lead to useful suggestions will need this basic info. As a new or first time user to HPLC, please help us by including it.

I see an increase in retention time of ~ 0.1 minute in your posted example. Is that all you are experiencing? If it is 1 minute change, now we have a serious problem (for your example), but not 0.1 minute.

It is unclear from your post if your HPLC system has a mixer installed. Please check. A mixer, even a static one is needed to mix solvents well. Mixing problems alone should not result in a predictable, positive change in Rt (as John C stated).

Thermo Accela pumps have vacuum degasser modules which are known to fail after ~ 4 years of normal use. How old is your integrated degasser and when was it professionally serviced last (Note; Many people forget to have these serviced and most thirty party service providers do not service them at all, but will stick a label on them that they have been "calibrated"). Poor quality mobile phase degassing is another common cause of retention time instability.

For most cases of observed retention time change in a predictably, positive direction, run-after-run, the two main causes are:

• (1) Column fouling. The column is fouled with sample (or buffers, matrix, additives). Replace the column with a fresh column and/or use a properly developed column wash method after each analysis to remove retained material from the column, then re-equilibrate for a ling enough time that it is stable and ready for another injection.
• (2) Inadequate column re-equilibration.

Here is a link to a free article on, "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

John R Carney Bruce Neagle Thanks! I'm new to this system, I'm most comfortable with Waters. From what I understand, the solvents are mixed with the proportioning valves at the quaternary inlet module, and the "dynamic mixer" is more for complex mixing and is optional. Our solvents are 99:1 H2O:ACN and 1:99 H2O:ACN, both have 0.1% formic acid. We largely run simple MeOH extracts of plant samples. I think the dynamic mixer is bypassed because it's not really necessary for us.

Perhaps it is a B-line pumping problem and simply needs new proportioning valves? Since we usually only use two lines, I was wondering if I could just plug one or two with faulty valves?

Thanks again. I've only been reading about it and will start troubleshooting with the machine soon.

And you still have not provided any information about the method or answered the most basic questions...

Yes, some mixing is done whenever two lines intersect, as in the proportioning module (which has four valves and acts as a gate, allowing different proportions in at a time or inside the pump head), but most HPLC systems have an addition mixer. *For the Accela, they have in internal inlet mixer plus the pulse dampener also acts as a mixer too. Optionally (or std on some), a dynamic mixer can be installed too. However, as noted earlier increasing retention times are NOT an indication of a mixing problem.

Using only two of the four inlet lines can indeed result in problems. The two unused channels dry up inside and the valves can leak (resulting in cross contamination). Do not plug the unused channels. Instead, double up on each solvent line and place each of the unused lines into the complementary MP bottles (same as you use). Flush each one with MP to purge them fully. Then go back to using your two lines. Periodically re-flush each of those lines (~ 2 days) to maintain the degassed liquid inside them.

If you would be so kind to share your actual running conditions, column dimensions (basic HPLC info) and also provide numbers (Rt's) of some of the significant peaks for say, 6 runs in a row, then we could provide some free insight.

The manufacturer supplies specifications for your proportioning valves, though I doubt they are responsible for your small drifting in retention times (as a column dies the RT will run to the solvent front). Rather, are you consuming any Formic Acid in your mobile phases by using 'alkaline methanol' in your extraction procedure?