Chromatography Forum

Hello! I have been having an issue with large, regular peaks appearing in my HPLC runs that have been making the instrument unusable. In short, when running gradient runs on the instrument (Waters 2535 Quaternary Gradient Module), I get large peaks that increase in frequency and intensity as the gradient increases. This problem is constant and occurs when using any of the 4 columns we have.
Below is an example run (50-100% ACN, 28min) with the following parameters:
A: H2O, 0.1%TFA B: ACN, 0.1%TFA
C18 or C8 column, 4.6x100mm
flow: 1mL/min.
T A B
0 90 10
1 50 50
12 0 100
20 0 100
21 90 10
28 90 10
https://imgur.com/a/tFqO7T5

-There are no obvious pressure spikes that co-occur with the peaks in the spectrum. The pressure profile itself is quite consistent; a sawtooth pattern with a maximum delta of ~35.
-The issue is also present when using methanol, but to a lesser degree.
-Varying the flow rate only changes the spacing between peaks and when the first peak 'elutes'

I assume the issue is something like a bad pump seal, but I am just trying to make sure before we shell out the money for an expensive repair kit. It could also be an issue in the gradient proportioning valve, but I don't know how to distinguish between that and a high-pressure pump issue. I do not think this is a contamination issue. The peaks are far too regular and consistently eluting. Their absorbance intensity shows no decrease across runs.

Things I have tried:
-Remaking solvents fresh daily, from multiple different HPLC-grade solvent containers. No change.
-Flushing the entire system with IPA. No change.
-Switching lines. Normally we use lines A and B. I switched to C and D and saw a very minor improvement, but the issue was still there.
-No injection. No change
-No column. Inconclusive, we didn't have the correct fitting to create sufficient backpressure.

Any help would be appreciated!

Hi Aaron,

I'm an HPLC noob to so take this with a grain of salt, but I believe that you're right on the money with your estimation that there is an intrusion of air somewhere in your system. If you flushed with IPA, then I would suspect if it was an air-bubble trapped on your PDA it would have been flushed out already. However, it does seem like no-matter what you try this issue continues to manifest and it suggests that the air bubbles are regular (like with every pump stroke of one of your pumps). The intensity seems to change as you move deeper into the method, and I'm not sure if it correlates with a higher organic concentration or what, but I would suspect that the pump you use for your organic has either a decayed seal that introduces air upon retraction, or potentially (if you have a degasser) it is not doing it's job correctly (but I doubt that this is the cause because of how regular your peaks seem to be eluting). Definitely wait for some more input for the real experts on the forum, but I suspect that there is air being introduced somewhere in your system.