Chromatography Forum

Hello all,

I have a question about baseline drift.

We have a method for determining protein concentration, and we use Waters H-Class system and Poros A20 column 2.1 x 30 mm for this method.
This method has a very dramatic Mobile phase gradient as below:

0.00 to 5.00 min: 100% Mobile phase A
5.00 to 5.01 min: 100% Mobile phase A to 100% Mobile phase B
5.01 to 10.00 min: 100% Mobile phase B
10.00 to 10.01 min: 100% Mobile phase B to 100% Mobile phase A
10.01 to 15.00 min: 100% Mobile phase A

Where,
Mobile phase A: PBS solution with pH 7.3
Mobile phase B: PBS solution with pH 2.5 (pH is adjusted with 85% phosphoric acid)

The baseline has a shape of hat with wide flat brim because of the dramatically changing gradient (like this, _Π_; I'm sorry for my poor explanation..). Fortunately, the drift (crown of the hat) is usually very small so that it does not affect on data processing and test result.
However, sometimes, the drift (crown of the hat) was increased more than about 10 times of usual baseline (like a toy soldier's hat, I'm sorry for my poor explanation again..), and we couldn’t find the root cause of this problem.
What could cause this huge drift?

The source for preparation of MP A and B is same, and we tried replacing mobile phase/column and washing the system to solve this problem, but it was useless.

Any suggestion will be greatly appreciated.
Thank you very much for reading my question!!

As you didn't mention what type of detector was used (DAD?), it's hard to speculate what causes the baseline problem? Under what circumstances do you see the problem? A new batch of mobile phase? If that's the case, it may has something to do with how phosphoric acid is added.

Thank you for your reply!
We use Waters TUV detector (280 nm).
At first, I also thought it was due to mobile phases.
However, sometimes, the same problem occurs when using mobile phases that have already been verified through previous tests (within due date).
We usually use a disposable pipette for adding phosphoric acid to the mobile phase B, and the problem arise when stabilizing equipment and preparing a new test. We tried many things to solve this problem (replacing Equipment/Column/Mobile phases, increasing stabilization time and washing equipment overnight), but it did not get better.
In some cases, the problem suddenly disappears when we try again after 1 or 2 weeks, so I could not find the cause.
I think there may be some mistake not noticed (or mechanical problems).

Is the problem observed when same batch of mobile phase B is used? Because the baseline drift occurs when switching from mobile phase A to B, I would run mobile phase B only with and without column and monitor the baseline . This test can determine if the problem is related to column and/or mobile phase B.

Thank you for your time and consideration!
Yes, the problem is observed when same batch of mobile phase B is used.
I tried to run MP A and MP B without column, however there was no evidence that the column is the cause of the problem.
I will try again using the mobile phase B only with and without the column according to your advice.
Thank you!

Hi!

I have the exact same problem, which has been more than a year...

Did you find a solution to this baseline problem? I am still troubleshooting...