cleaning column (UPLC)

[catlover]

Hi everyone,

I would like to clean/wash the 1.7 um BEH column because there are strong peaks in MRM mode of blank solution.
My target analyte is OP pesticide, and I'm strongly sure that the MRM mode is correct.
I tried a run bypassing the column and those strong peaks disappeared. Therefore, I guess the column is contaminated with pesticide.
However, I don't know what is the best way to clean the column.
I tried as told to run the column overnight with 90%ACN :10% H2O in 0.4uL/min.
However, the noise are still there.
Then, I run the gradient several times, and it seems get better (but still very strong peak in 10^4 and 10^5).
I would like to know if clean the column with only one run for overnight, or clean it with many runs for few minutes are better.
Please kindly advise if you know, thank you.

[Gerhard Kratz]

If you get these strong peaks everytime you do a blank injection please check your injection system. Are your samples dissolved in a different solvent or solvent composission than your mobile phase?

[HPLCaddict]

Gerhard already had a good point.
Before trying anything else including time consuming column washing procedures I'd swap that column with a brand new one or, if you don't have a new one at hand, with a different one. Just to make sure that it's indeed a column contamination and not your system our your eluents.

[catlover]

If you get these strong peaks everytime you do a blank injection please check your injection system. Are your samples dissolved in a different solvent or solvent composission than your mobile phase?

Thank you for your reply.
As the mobile phase run in gradient mode, I change the solvent to match the initial state of gradient mode.
I tried zero injection and peaks are still there, so I guess the source is not the injection.

I also tried infusion mode of all mobile phase and still there is no peak. But when flowing with elution bypass the column, it comes the normal background noise (no more peaks). Thus, I think the source of contamination should be column.

By the way, I would like to know if gradient mode would cause high peaks because I also tried the isocratic run with 90% ACN and there are no peaks coming out......
It seems those strong peaks coming only when running gradient mode (I would confirm it later).

Gerhard already had a good point.
Before trying anything else including time consuming column washing procedures I'd swap that column with a brand new one or, if you don't have a new one at hand, with a different one. Just to make sure that it's indeed a column contamination and not your system our your eluents.

We buy a new one and would see if the source really come from the column.
Still I am curious to find it out if there is any other ways to solve this problem.
Thank you for your respond~!

[HPLCaddict]

Sounds as this could also be the old problem of contaminants in your A eluent...do the peaks get larger when you increase the equillibration time between two gradient runs?

[catlover]

Sounds as this could also be the old problem of contaminants in your A eluent...do the peaks get larger when you increase the equillibration time between two gradient runs?

Thank you for your respond.
It really happen when the composition of ACN increased in MRM mode.
As I remember, the gradient is set as follow:
0 min, 90% buffer, 10% ACN (actually it is 0.1% acetic acid in ACN)
1 min, 90% buffer, 10% ACN
4 min, 5% buffer, 95% ACN
5 min, 5% buffer, 95% ACN
7 min, 90% buffer, 10% ACN
8 min, 90% buffer, 10% ACN

The curve is linear slope, and the buffer is 0.1% acetic acid in 10 mM ammonium acetate.
Although I observed this, I have no idea of why it could happen...
Would you kindly explain if you know? Thank you.

[E11022]

For UPLC, i am not sure as i am using GC.

If GC, i had the same problems before. i try heating up the oven by 5 - 10 degree celcius

If your column is contaminated/saturated, increasing the oven temp might remove those unwanted contaminants.

Hope this help.

[HPLCaddict]

While GC and HPLC share some characteristics, you will not get rid of column contaminants just by heating the column. Increasing the temperature while flushing the column might help, though.

What I was originally aiming at is that this might be indeed not a contamination of the column or the system, but a contaminant in your mobile phase (aka gradient ghost peaks). In your gradient, you have a reequillibration step of 1 minute at the end (7-8 min 90%A,10%B). Run several blank gradients (i.e. no injections) until those interferring peaks are stable. Directly afterwards, run several blank gradients with an increased equillibration step (let's say 7-10 min 90%A, 10%B). Do those interferring peaks increase in size or do they stay the same with increased reequillibration?

[catlover]

While GC and HPLC share some characteristics, you will not get rid of column contaminants just by heating the column. Increasing the temperature while flushing the column might help, though.

What I was originally aiming at is that this might be indeed not a contamination of the column or the system, but a contaminant in your mobile phase (aka gradient ghost peaks). In your gradient, you have a reequillibration step of 1 minute at the end (7-8 min 90%A,10%B). Run several blank gradients (i.e. no injections) until those interferring peaks are stable. Directly afterwards, run several blank gradients with an increased equillibration step (let's say 7-10 min 90%A, 10%B). Do those interferring peaks increase in size or do they stay the same with increased reequillibration?

I run several blank gradients and the peaks intensity drops to nearly background.
It is my fault not to flush the column after daily runs. I would see if the problems comes again.
Thank you for your respond.