Chromatography Forum

Hello
I have some problems with my LC system and the pressure.

Usually I use two different mobile phases (H2O, MeOH with NH4-Ac and Na-Ac) in gradient mode and therefore I have a pressure around 1300psi.

Nowadays, the pressure has increased up to 1700psi in the beginning and during the sample runs the pressure increased slightly within the time. I know maybe there is some dirt etc., but I cleaned everything with backflusing the column (also change the column), change the mobile phases, and so on.

I also observed some strange things, I guess. When i just use the water phase the pressure is nice around 900psi, and when I introduce the MeOH the pressure increased. I know during mixing the pressure will change, but when I just use the MeOH, I would expect to get a lower pressure for this (is it like that?), but it is also up to 1400psi.

I don't know what I can do anymore!

By the way, during backflushing the column the pressure is lower than in real direction, it should be the same, shouldn't it?

Thank you for your suggestions!

Mikro

With 100% methanol, the pressure will always be lower than with water. Methanol/water mixtures can have higher viscosity than with water or methanol alone and hence show higher pressure with LC. The pressure should be highest around 50:50 methanol:water. You can look up a viscosity diagram of methanol / water mixture to see what I mean.

Sometimes backflushing the column will not be able to remove all particles or contaminants in the column (also can damage some columns). I highly recommend using a guard column to protect your analytical column. We use a guard column and also a precolumn filter before the guard column. That way you can just change the relatively inexpensive filter frit or guard column instead of changing the analytical column.

You might also want to change the pump frit. LC pumps usually have a frit which can get clogged with pieces of seal and mobile phase gunk over time.

ok, so I was right with the pressure for the methanol. Good to know.

But why is the pressure so much higher with MeOH, actually in both pumps. And as I said, I changed the precolumn and also the analytical column to test it, and it was the same!

And what do you mean with the pump frit?

Thanks!

Mikro

Could it be that the high MeOH pressure was obtained when there was still water in the system??

I guess not, I have waited up to 1 hour before I checked the pressure. This morning it was the same, pure water (1000 psi) after introduction of methanol pressure goes up to 1500psi. Of course there will be an increase because of the mixture, but probably not that much. As I mentioned above, usually we have a pressure of 1300 psi!

So I think, something happened when the methanol is in the system, also, when it is "alone" there!

Hi mikro,
Is your methanol definitely methanol - not someone elses labelled bottle or a bottle someone else has filled?
WK

yes, it is a new bottle of HPLC grade MeOH!

Just an unlikely thought: If someone has been using anything salty in your system, you can get pressure problems on using pure methanol because many salts have limited solubility in pure methanol.

We had big problems some years ago with a Waters Alliance system that was used for ion exchange chromatography with strong phosphate buffers, and also for reverse phase chromatography. The reverse phase always caused the trouble, because even after washing lines according to Waters' instructions, sufficient salt remained in the inlet filters that putting them in 100% methanol led to blockages. Normally we got stuck check-valves, but whether you get stuck check-valves or other forms of blockages will depend on your salt and your pump design.

Mikro,
Are you using the same line in for all these runs or is the methanol on a different line? Blocked Solvent filter?
WK

hello,
it is more or less the same line, I mean I have two lines in the different "buffers", a T-piece and afterward 1 line.
But, I think it is not a blocked line, because without a column, I just have a pressure of 45 psi, I guess it is because of the different diameters during the flow line! Isn't it?

So when I'm reading this, maybe the reason stuck in the column itself?

Do you think the compressibility settings in the pump can be responsible for that phenomenon?

I checked the settings and there are the same settings in both pumps (I guess for water), maybe since the technician was here a few weeks ago. This shouldn't be the case since I use one of them with MeOH, shouldn't it?

Thank's!

Mikro

Mikro,

1. Is your system a binary or quaternary pump?
2. If you tried using MeOH without actetate, is the problem still there?
3. If your pump is Agilent 1100/1200/1050, release the purge valve on the pump head, run 100% H2O with flow rate 3ml/min. If the pressure is >10 bar, there is probably the PTFE frit inside blocked. (usually I have 0-1 bar when the purge value is released)
4. Did you swap the solvent channels and have the same result?

Mikro, my guess is that the high pressure is due to the precipitation of the salts in your UPLC system.

Try connecting a new column just to check if the problem persists.

Also 45 psi without a column is high pressure as far as i know, although I don't now what make your LC is. The pumps I am familiar to display 0 psi when no column is connected, so there's probably salt stuck in your check valves, piston seals etc.

I would perform disasembling and cleaning of the pump, and flush the system with 100% water for about an hour.