Pressure Buildup

[Pierre Robitaille]

Hi all,

I have been having some increasing pressure problems and was wondering if anyone might have an idea as to why. I am using a Symmetry Shields RP18 19x300mm column with a flow rate of 5ml/min using methanol and water + 0,1% TFA (1:1) as eluent in isocratic mode. I noticed that as the separation was proceeding the pressure would increase and ultimately reach the pump limit (400 bars). I decided to flush the column by gradually going to 100% water to 100% acetonitrile to 100% tetrahydrofuran and then reversing the process. After going back to my analysis conditions and running an analysis the pressure keeps building up and reach the pump limit before the analysis is complete. Anyone has an idea what the problem might be?

Thanks in advance for any help.

Regards, Pierre Robitaille

[Koen Hollebekkers]

Pierre,

My first impression is that you have a blocked column frit at the front of the column. Are you using a guard column?
First of all, you have to check the system with your SST column. In case you don’t have one, use a column that you know for 100% sure that’s working well. Than test the performance.
In case that this column also shows this failure, possibly the inlet frits in your system are blocked.
In case this column is working fine, you have to contact your column supplier and inform them. If your lucky they will repair it.
Let me know if this solved the problem.

[Koen Hollebekkers]

Two other commends;
· Why are you using such a high flow (H/U curve!!)
· What kind of application are you doing;
· I would not recommend to use 100% THF (It will dissolve peek)

[Mark Tracy]

The comment about 100% THF is quite accurate. PEEK swells and cracks on exposure to pure THF; if a PEEK capillary can't expand outward it will expand inward and close off.

The flow rate of 5 mL/min is actually quite low for a 19 mm diameter column, and even with the relatively high viscosity of methanol/water should not cause trouble.

Perhaps this is sample related. What is the analyte (in vague, general terms) and what is it dissolved in?

[syx]

· I would not recommend to use 100% THF (It will dissolve peek)

How much is the recommended dosage of THF that could be used for column cleaning procedure?
We have found procedure for reversed phase column as follow:
Rinse with 10 column volume each of:
95% water/5% acetonitrile (for buffer removal)
THF
95% acetonitrile/5% water

Regards,
Siswanto Tanuatmojo

[Uwe Neue]

You should have more pleasure and less pressure...

Assuming that you are using a 5 micron packing, your pressure should be in the order of 50 bar, not 400 bar. My first question is the nature of the tubing that connects the pump to the column and the column to the detector. Replace the column with either a direct connection between the column inlet tubing and the detector inlet tubing or a short piece of 1 mm i.d. tubing and run the gradient again with this set-up. Let us know what the pressure is without the column.

If the pressure is substantial, then let us work on replacing the tubings in your system.

We can then explore what to do with the column.

[HW Mueller]

If I read Pierre´s question correctly then he does not have a problem due to the THF, but rather a resistance due to the injected sample. I didn´t answer earlier, because I am tired of asking a return question, but here it is: What are you injecting on the column? Maybe you need a pre-cleaning.

[Koen Hollebekkers]

I'm not totally sure of it, but out of my bare head I remember that the max is 80%.
This will not solve your problem. Like Uwe mentioned earlier. Check the system without column. And try another column with good performance. Otherwise it will be guessing what the problem might be (column or system).

Good luck!!

[Pierre Robitaille]

Thanks everyone for the suggestions.

Koen, you were partly right. It is the frit in the purge valve that was completely blocked, it was black! So after changing it, the pressure came back to normal levels. I guess filtering your solvents still lets small impurities go through.

Mark, I will be more careful next time with THF. I was under the impression that PEEK tubing was rather inert to organic solvents, thanks

HW, I am injecting a modified amino acid that is easily solubilized in the mobile phase and I also filter the sample prior to injection.

The HPLC we have is rather new and I didn't expect the frits to be that dirty so soon. Next time the pressure starts building up again, this frit will be the first thing I'll be checking.

Again, thanks to everyone for your time. It is highly appreciated.

Best Regards, Pierre Robitaille

[Koen Hollebekkers]

Hey Pierre,

This is well known problem, I also suffered many times this kind of problems. I hope that you see now how important a SST procedure is. You can differentiate immediately if the problem is related to system or column.
By the way what kind of equipment are you using?

[Pierre Robitaille]

Hi Koen,

I am using an Agilent system with a fraction collector and semi-prep pump. We use it mainly to separate compounds that we can't separate by flash chromatography.