It is likely this carryover is from the injected samples that have been selectively retained and need a stronger organic solvent to 'boot' them off the column.  So instead I would try as your Mobile Phase B H2O: ACN: Isopropanol (25: 25: 50 V/V) +0.1% HCOOH (we don't want a pH gradient).

I am not sure at all, but do You wash Your needle/Injector? with our systems we wash those routinely 3 times before and after the injections with pure solvent.

However if You have that much left in the loop/needle it smells like a major part of your sample is not getting into the main column....

If You have alread tried to go up to 100% ACCN without effect I do not believe the compound is on the main column...

Hi Phil.

I think you could switch auto injector valve( for by pass/ inject) for remove possible analyte residue.This is very easy configurable in Analyst software. I had  same problem with similar equipament in my work. Some times loop remained analyte residue in your loop injection. You could try wash needle with a solvent in wich your analyte is more soluble.

Regards

The sample injection protocol in Agilent 1290 bypasses the needle for a few seconds only for drawing the sample and then switches back to the mainpass position, in which the mobile phase always continuously flow through the needle to the column, for minimizing carryover. So, there is a little chance of carryover from needle internal.

A couple of things. One: have you reduced the injection volume or better yet diluted the sample? Two: I have an Agilent QTOF in which I experienced the sample attaching itself to the plastic lines.... Lastly have you varied the gradient to have more organic in the initial steps?

use your mobile phase for Rinse solvent:.

Here's my few cents, if it is the autosampler issue, then why he did not have issue with isocratic mode? I assume that the autosampler program (no of rinse and type of solvent is the same). I had this issue before (see my research gate on the question that I asked). The solution that I had is from an engineer, not chemist, from Shimadzu. The column has some imperfection and retains your analyte. You can test by programming as usual but after you equilibrate the column at 8 min, bumping the mobile phase to 80% B immediately for 5 min and dropping to 10% B immediately and staying for 5 min. Repeat the 80% B again and 10% B again one more time and still collecting the data. If you column is good, you should not see the analyte peak after the two cycle because it should be eluted at the first gradient. However, if you see two more peaks with lower response because of the carry-over, it means you need another column that will not give you that. Try this test and tell me the result.

I don't see a cycle time to re-equilibrate to 90:10 water:ACN. Did I miss it? It will take some time to go from 10:90 to 90:10; I'd guess 5-7 minutes at least, depending on your flow rate (you didn't give us flows or column dimensions). if you don't properly re-equilibrate the column you can get all kinds of wacky things occurring.

Thanks for all your input!

In the end it turned out to be very strong carry-over due to the physical/chemical properties of the analyte. The analyte showed very strong affinity to virtually all column phases, which means residual amounts remained stuck on the solid phase of the column which kept on eluting in small fractions with each subsequent blank run. I have found a highly deactivated C18 column that showed minimal carry-over with which the analysis could be adequately performed.

Hi Phil.

Carryover is two types one is system carryover while another is column carryover. If carryover comes from column than change the column. If your compound has high affinity to your stationary phase it will take more time for complete elution so better the change the column chemistry to resolve the problem. If carryover comes from system than increase the rinsing volume, rinse dip time, change the rinsing programme, change the composition of rinsing solvent( rinsing solvent must be dissolve your compound).

Next time you have a carryover issue, please read this article to troubleshoot and understand the reason for it. Better yet, read it and avoid having the problem at all.

"Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems"

https://hplctips.blogspot.com/2015/02/carry-over-carryover-contamination-in.html

One additional comment on the above method.

For most HPLC, LC-MS or LC-MS-MS methods where acetic acid is used, you may want to substitute HPLC grade (or better) formic acid (0.1% Formic acid is a good starting concentration for many methods). Acetic acid is very "dirty" and impure, even in the higher grades found in most labs. It also degrades quickly, often turning brown. It has been my experience that these types of changes add a great deal of noise and artifact to the baseline during analysis which are not welcome. *I have improved the quality of many methods and solved many noise or artifact problems in client systems by substituting formic acid for acetic acid.

High quality formic acid does away with many of the above problems and also improves ion formation. Additionally, formic acid's lower pKa allows either lower volumes to be used (vs. acetic acid) or lower pH values to be obtained. BTW: This seems like a good time to remind everyone that when running LC-MS analysis, please do not substitute or use TFA in your mobile phase unless you wish to deal with the resulting long term contamination of your MS source that results.