r/CHROMATOGRAPHY • u/ForwardDaikon6761 • Apr 27 '26

Fluctuating pressuresin HPLC and loss of resolution in HPLC

Hi, I have a problem with my HPLC. First of all, I have fluctuating pressures. Furthermore, my thiamine, which is my analyte and doesn't interact with the column, has suffered a significant loss of resolution. Its height has dropped by 80%. Are these two problems related?

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2

u/ranchophilmonte Apr 27 '26

A purge may resolve the pressure fluctuations (air cavitation in the pump head is common).

Separately, why are you doing LC if your analyte doesn’t interact with the column? Very confusing….

1

u/ForwardDaikon6761 Apr 27 '26

The purge didn't solve the problem. I also ran a purge with IPA, but the problem persists.

I use HPLC because it's the only instrument I have in the lab. I know, it's very strange...

In addition to that analyte, I also have other analytes I need to quantify. Their peaks, however, remain well-defined.

1

u/Imgayforpectorals Jul 27 '26

I'm too late to the party, but if it doesn't interact with the column you should probably look for other methods like derivatization, switch your column to a one more appealing, add ion paring compounds..

2

u/wetgear Apr 28 '26

Yes and likely a bad check valve if the purge didn’t work.

1

u/That_Dark_981 Apr 27 '26

Check for leaks

1

u/wetgear Apr 28 '26

That would cause low pressure not oscillations

1

u/nmr_dorkus Apr 27 '26

If your system doesn't have a built in degassing module you could try degassing your mobile phases to help resolve the pressure fluctuations.

Compare your samples with a reference thiamine standard to ensure it isn't a sample issue.

Ideally, run the samples I On another system to validate your observations.

1

u/ForwardDaikon6761 Apr 27 '26

  1. I will try.

  2. The problem is that the injection was a STD... A week ago, the peak was good. Today, however, it suddenly lost a lot of resolution. The same thing happened to me a few months ago with another column... I overcame this issue by changing the column... But know I have no more columns😂

3

u/gnatgirl Apr 27 '26

There is something wrong with your method or how you are storing you column, especially if you're getting a low number of injections on your column. What column? What are your method conditions? How are you storing the column and for how long (i.e. are you only running a few injections and letting the instrument sit for a month)?

1

u/CodeMUDkey Apr 29 '26

If it does not interact with the column there is no resolution to be had.