Chromatography Forum

We are doing LC/MS method development using ABISCIEC 5500 MS with the Agilent 1200 series LC unit.

We are having issues with carryover and we have narrowed it down to the Agilent auto sampler. We believe the problem is leftover/residue in or around the needle. We will run two run blanks (40 ul) that show no peaks, then run our sample with the peak we are looking for, then another set of run blanks that show carryover with the peak from our sample. This peak gradually lowers in intensity with each run blank.

We have tried running the automatic needle wash/port using 20/80 acetic acid/methanol as the wash in the port. If I'm correct, this is supposed to clean the outside of the needle only. I have also tried running an extra line in the injector program to draw/eject this same wash before running the sample with no luck. This was done in an attempt to clean and rinse out the inside of the needle. I have also tried different combination of washes using methanol/water/acids/run blank solution.

The method we are running is a 20 minute gradient elution with A = water with 0.1% formic acid, B= acetonitrile with 0.1% formic acid.

I was hoping that this forum could provide some suggestions or any troubleshooting tips and hints. I would really appreciate the help.

Thank you.

The needle wash only washes the outside of the needle because the inside of the injection needle is constantly being washed by mobilephase in all but the bypass steps.

Thanks for clarifying that for me.

Perhaps it's my choice of wash? I'm using mainly polar washes.

Maybe a nonpolar wash will be better? Acetone or ethyl acetate. But I'm worried about wearing out the tubing.

Also is there any advantage in adding a line in the custom injector program to do a draw/eject, for example to draw from a vial with a wash solution (Vial 1) and then eject into another vial (Vial 2)? Or is this step redundant with the automatic wash and since the MP is washing the needled in the mainpass position?

Thank you.

Are you sure your sample carryover isn't coming from the valve/rotor seal? If I remember correctly, there is a valve switching/cleaning operation in the Chemstation B.04 software for the 1290 well plate sampler that lets you flip the valve from load to inject a specified number of times after the injection in cases where the rotor seal is suspected of causing carryover. I believe in older Chemstation versions you can create a line in the autosampler program to switch the valve at predetermined times in the run. Maybe you need to have the valve flip near the end of the run, so that all parts of the rotor seal "see" the high percentage of acetonitrile, to better wash the valve? Otherwise, you can try 100% acetonitrile or 0.1% formic in acetonitrile instead of 20/80 acetic acid/methanol as the wash solvent, or try washing for a longer period of time (20-30 seconds), or both.

Are you by any chance utilizing the overlapped injection option? My lab had a significant carryover problem with one method using an Agilent 1290 autosampler. We were trying to increase throughput by overlapping injections. This however was interfering with the needle rinse features when the autosampler switched from main pass to bypass. When we stopped overlapping injections the carryover completely disappeared.
Just a though.
-Chemboy