Peak shoulder on all peaks

[catlover]

Hi everyone,

I prepared fresh mobile phase after discover this problem. But the problem is still there.
I guess it may caused by dirty guard column but I have no one to change.....
Is it possible that lowering injection volume or increasing flow rate can "help" giving a shape peak?
What else I can make it better?

Here are my setting on pesticide test (LCMSMS), I would be grateful for you sharing. Thank you.
Column: UPLC BEH C18, 2.1mm x 50mm, 1.7 um
Injection volume: 30 uL
Flow rate:0.4mL/min
Gradient:

0 min, 90% buffer, 10% ACN (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

Buffer is 0.1% acetic acid in 10 mM ammonium acetate.


I also tried a new gradient that give higher intensity peak but still there is peak shoulder problem.
Gradient:

0 min, 90% buffer, 10% ACN (0.1% acetic acid in ACN)
1 min, 90% buffer, 10% ACN
3 min, 20% buffer, 80% ACN
4 min, 20% buffer, 80% ACN
5 min, 10% buffer, 90% ACN
6 min, 10% buffer, 90% ACN
7 min, 90% buffer, 10% ACN
10 min, 90% buffer, 10% ACN

[Gerhard Kratz]

Peak sholder is mainly caused by void volume in your guard and/or analytical column. If you don't have a replacement guard column take the guard out of your System for one injection with Standards. Than you can see if you still have shoulders on your Peaks. If so, than order a new guard and a new analytical column. You can try to rinse your column and than built it in with revered flow without connetcing to the detector. Rinse again and than connect the detector. That might help for a few injections, one day or so, but not longer. Sorry for These bad News.

[HPLCaddict]

Did this method work in the past or did you try it for the first time?
If you've never used it before, it might be as well an inherent problem of the method itself and not a column problem. First thing that comes to my mind is that 30µL is a huuuuuge injection volume for such a tiny column (a 50x2.1mm column has a dead volume of only ~110µL!). Maybe it's just volume overload? You might get away with high injection volumes if your solvent has much lower elent strength than your mobile phase, but since your gradient starts at only 10% organic, your solvent cannot be much lower...

[Consumer Products Guy]

If I get bad peak shapes, first thing I investigate is the shape of a smaller injection volume.

Yes, I agree that 30µL is a huge injection for that dimension of column, especially when the initial % organic in the mobile phase is so low. When we used 2.1mm x 100mm columns, we used injections of 5 µL.

[catlover]

I checked previous chromatograms of 30 uL injection, those peaks are very nice even with a high injection volume.
I traced back and discover that the problem started after injections of recovery and sample.
So could the problem caused by column?

[tom jupille]

When *all* the peaks on a chromatogram show the *same* problem, the chances are that the problem occurred before the peaks had started to separate (i.e. at the column inlet). In my experience this has most often been the result of an inlet flow profile anomaly:
- an air bubble trapped at the column inlet (air bubbles will redissolve, so if the problem comes and goes, this is the most likely)
- a partially plugged inlet frit (this usually has an abrupt onset and once it happens, it is consistent. Iif you review the chromatograms, you can often tell which sample caused the problem).
- a headspace at the column inlet (this often -- but not always -- develops gradually).

The "strong diluent effect" can also distort all the peaks, but usually not the *same* distortion on all of them.

If you are using a guard column, change it (that's what guard columns are for!).

[catlover]

Hi everyone~!
The problem was solved!!!
First of all, the problem doesn't related to the injection volume because the problem remain after reduce the injection volume.
Then I wash the column with 1:1 ACN:H2O, the shoulder seems reduce.
After that, I back flush the column about an hour with 1:1 ACN:H2O. Finally, the sharp peak shape comes back~!
You all are right that the problem caused from the column inlet.

I also order a guard column for protection of column.
Anyways, thank you very much for your suggestion. This help me a lot