High pressure help needed

[Cardinal Fang]

On the Metadrenalines method I'm running on our Shimadzu LC system, I'm getting high pressure. It normally runs at about 130 bars. We don't get any peaks when we try to put samples through.

Started off at 280 bars. I've tried tracing the tubes back sequentially to see if there's a blockage, but that didn't change the pressure.
We initially thought it was the column, but replacing the column with an old one we knew wasn't blocked didn't so anything either - the pressure was still far higher than it should be. We also changed the prefilter in case that was clogged, and also connected in the column without the prefilter, which also didn't affect the pressure.

Overnight we ran an IPA wash through the system, to clear any blockages. This brought the pressure down slightly to around 250 bars, but this is still too high. We tried rinsing the flow lines in case there was a blockage, but this hasn't made any difference.

I've also tried reverse rinsing the outlet tubing with IPA, as it suggests in the autosampler manual, but the pressure is still higher than it should be.

As we still wondered whether it was the column, I reversed and cleaned it, as per the manufacturer's instructions. This brought the pressure down to 190 bars.

Putting a brand new shiny column straight out of the box didn't do anything either. The pressure is still 190 bars. I know new columns can have varying pressures, but that make (Kinetex), the highest a new one has ever been new on our system with that method is 150 bars. But 130 - 140 is about where they normally are when new. 190 is abnormal.

Anyone have any ideas about where to go next? Everything is screaming there's a blockage somewhere, but I cannot for the life of me find it.

[Hollow]

I'm not familiar with the Shimadzu system, but have bypassed the Injector / Loop?
Proably the blockage is there, if the high pressure exists even without any column.

[James_Ball]

What is the pressure at operating flow with no columns installed?

[Cardinal Fang]

I'm not familiar with the Shimadzu system, but have bypassed the Injector / Loop?
Proably the blockage is there, if the high pressure exists even without any column.

When the column is removed the pressure drops to 14 bars. This is what made us think it was a clogged column. However the fact that we still get the same symptoms even with other columns (both an old one we know isn't clogged, and a new-out-of-the-box one) makes us reconsider that.

CF

[Multidimensional]

This is basic step-wise troubleshooting. Plain and simple, if after removing the HPLC column and replacing it with a zero dead volume fitting AND running the same mobile phase composition and flow rate results in the back pressure dropping down to a typical system pressure (e.g. 14 bars, however it could be almost any number depending on your HPLC config), then the HPLC COLUMN is what is contributing the most to the back pressure. By design, Columns are the primary restriction in an HPLC system. Be sure to run this basic check with the zero dead volume union while running the actual method. This includes injecting mobile phase (same volume) too as many clogs will occur in the autoinjector's flow path as that is the first constricted area the sample hits. Such a basic test will point to the obstructed area and allow you to solve the problem.

[James_Ball]

This is basic step-wise troubleshooting. Plain and simple, if after removing the HPLC column and replacing it with a zero dead volume fitting AND running the same mobile phase composition and flow rate results in the back pressure dropping down to a typical system pressure (e.g. 14 bars, however it could be almost any number depending on your HPLC config), then the HPLC COLUMN is what is contributing the most to the back pressure. By design, Columns are the primary restriction in an HPLC system. Be sure to run this basic check with the zero dead volume union while running the actual method. This includes injecting mobile phase (same volume) too as many clogs will occur in the autoinjector's flow path as that is the first constricted area the sample hits. Such a basic test will point to the obstructed area and allow you to solve the problem.

To add a little to this(good advice as always), if I remove the column and the pressure remains high, I then work backwards towards the pump, opening each connection in line going back to the pump. When you find the one where the pressure drops then you know the clog is past that connection and before the next one.

This problem above does seem to be the column, but it may not be bad. Especially if it is an older pump, it could be the pressure sensor has drifted a little higher.