Interesting pressure phenomenon

[Zoraku]

I just recently ran a sample at a 6 month time point for a stability storage data. I noticed that after the first 10 injections the pressure was slowly starting to increase after every injection. Eventually towards the end of the run, I couldn't run a sample due to the pressure being too high.

My mobile phase is a gradient of Water/ACN with the following parameters
Time %Water %ACN
0.5 70 30
6.0 50 50
5.0 30 70
2.0 50 50
2.0 70 30

The problem is when the mobile phase composition becomes 70/30. This is when the pressure is at the highest (which makes sense as this combination of solvents should have the highest viscosity out of all of the ratios during the gradient). The problem is that anytime I did anything to reduce the pressure such as rinsing the system with pure organic solvents, etc... as soon as I started to equilibrate the system at that 70/30 ratio, the pressure slowly rose the entire time until it was erroring at too high of a pressure - even with no injection.

So I swapped the mobile phases to different lines - maybe something was wrong with a specific line. No fix
I removed the column still have about 250 bar of pressure. I tried to purge all the lines, but again, when I run 70/30 the pressure increases now back up to about 150bar.

It seems that the only time the pressure doesn't continually increase is if I'm at 50/50 water/ACN or lower.

The interesting thing is that even with this increase in pressure, my results at 6 months are nearly identical to those of day 0, including peak shapes and retention times. So it didn't seem to have a negative impact on the run.

I'm not very familiar with perkinelmer instruments mechanically - so I'm hesitant at the moment to start taking apart the pistons and the pump unless I have an idea for what I might need to be looking for. The instrument is a PerkinElmer Altus.

Sorry for the long post, just trying to be very detailed. Any suggestions would be great.

[Hollow]

may we ask for more detail about flow rate and column/particle dimensions first, before wild guessing starts?
also tubing diameter if known? 150 bar without column seems quite high to me, unless flow rate is very high.

What pressure did the method produce in the past?

[James_Ball]

I don't know anything about the PE instruments but the Agilent pumps have a filter at the outlet of the pump that catches the wear particles from the pump seals. When this filter gets clogged you will see similar problems with high pressure when column is disconnected. If you have a filter some where in line before the column or autosampler then that could be where the problem is.

[Zoraku]

I don't know anything about the PE instruments but the Agilent pumps have a filter at the outlet of the pump that catches the wear particles from the pump seals. When this filter gets clogged you will see similar problems with high pressure when column is disconnected. If you have a filter some where in line before the column or autosampler then that could be where the problem is.

Yes James, this is exactly what the issue turned out to be. I had never taken apart a PE HPLC so I was hesitant to start unscrewing connections and removing seals or frits. It turns out right in the front there is a filter. When I removed the filter and connected a union, my pressure immediately dropped. Even after sonicating the piece, it didn't help. So I will order a new one and replace it completely.

Problem solved! Thanks for your input.

[Zoraku]

So, to update this post, I still seem to be having an interesting pressure issue. I had thought I fixed the problem initially but now the problem seems to have changed.

Running at 1ml/min, with a mobile phase of 75/25 ACN/Water, I am seeing the pressure of the system continually increase at a slow rate over time. There are no injections, just merely equilibration of the column. I had just ran a column wash gradient yesterday, and now while running the mobile phase equilibration I am seeing this phenomenon. I'm not quite sure why the pressure is continually increasing.

The column is a 4.6x100mm 3u column. I should be seeing a pressure of about 130bar, which I did initially after changing the filter as mentioned in the above posts. But as I run the system the pressure just continues to increase. I'm curious if anyone has seen something like this, as I'm running out of ideas.

[Steve Reimer]

Next step is to find out where the increase in back pressure is occurring. Then figure out what is accumulating (bits of pump seal? mobile phase growth?)

[EmpowersBane]

Pressure issues are normally a blockage somewhere along the stream and as you are experiencing, it can creep up with an increase in Water:Organic mixtures for a variety of reasons. Is the quality of your water very high? Water with worn or old filters can accumulate a build up of organic "rubbish" which can gather at the head of your column and increase pressure.
The sinkers in your high aqueous line may be partially plugged, try replacing it. Or maybe disconnect each section of the pump then test for pressure: Disconnect the line from check valve to solvent selector, then disconnect from mixer, from column etc, testing at each stage. It may be your column as well, it could contain some strongly retained material which is blocking the steady flow of your sample molecules. Good luck!

[Zoraku]

I ended up replacing the water and that did the trick. While I am using high purity HPLC water, it had been sitting for about 3 months. I did not necessarily see any microbial growth but that doesn't mean it wasn't there. After I changed the water the pressure stabilized and the entire 60 injection run that I performed after changing the water had no significant pressure difference between the 1st and final injection.

Simple fix, thanks all.