rapidly climbing back pressure with new column

[c13hart]

Hello!
I am having an unusual problem with back pressure. My machine, an older Agilent 1100, is used heavily. I installed a new column (RP-18) and after only four or five days of use, the back pressure was way up (should be around 28bar, climbed to 50bar). The rest of the system has pressure that is just a little elevated (6bar or so) so I'm thinking that I have some seals or something breaking down that are contributing to small particles in the system and prematurely ruining my column. I installed another new column to test this and sure enough the pressure stabilized around 28 and a few hours later is up to 35. My mobile phase is isocratic methanol.

My real question: I have read the relevant trouble shooting guides, but I am looking for the opinion of an experienced user. Which seals should I replace first? I replaced the needle seat assembly and needle recently, and I do the purge valve PTFE frit regularly. Everything else has been in place for at least 1-2 years (I inherited this instrument in late 2012).

[tom jupille]

Normally I'm a proponent of "change one thing at a time", but in this case it looks like it's overdue for a full PM.

Of course, you could stick an in-line filter just upstream of the column (something on the order of 0.2 or 0.5 micron porosity), let it run for long enough to get a significant pressure increase, and then look at the filter frit under a magnifying glass or stereo microscope. The color and shape of any trapped particles shoud give you some insight as to the source.

All of that said, injector rotors are usually pretty robust; I'd probably look at piston seals as my first suspect.

[dhobbs]

In my experience, the purge valve frit catches any pump piston seal particles very effectively. The metering piston in the autosampler is essentially the same design and is downstream of the frit, so you may want to look at that seal. Another possible source could be bits of vial septum or particulate from your samples.

[Gerhard Kratz]

Do you use stainless steel tubing and have a ferrule from stainless steel - check if the outlet of the tubing is not smashed.
Second thing is to test your new column, just do the QC test if the column will show the same number of theoretical plates and RTs.

[danko]

If the column is the culprit, why changing seals etc. Remember the old saying: “If it ain’t broke, don’t fix it”
Now, how to identify the problem, you’d ask yourself: Does the pressure increase coincident with the number of injections? If yes – and it’s most common – then you have compatibility problems with the sample and the mobile phase. I use to observe a mixture of mobile phase and a realistic sample amount to make sure no precipitations occur, before injecting on system.
Maybe it a good starting point in your troubleshooting?
If number of injections does not correlate with the pressure rise, then it could be the mobile phase itself – i.e. one or more components aren’t completely dissolved.

Best Regards

[c13hart]

Good morning! Thank you all for the thoughtful advice!

Problems I can eliminate:
-I am sure it is not the mobile phase, this system is used only with HPLC grade methanol (Isocratic) with no dissolved salts or any other additives
-All of the samples run are first filtered through .22 micron and always have been, so I hope that is not the problem
-None of the SS ferrules appear smashed or damaged

Fixes I will attempt:
-pump pistons, which have not been replaced in the last two years
-metering piston from autosampler

I'm more interested in just getting things working, so I might recklessly fix everything at once and then test ; )

[danko]

Filtering the sample solutions guarantees in no way that the sample will remain in solution upon contact with the mobile phase.
A mental experiment here: You have a sample in aqueous solution where it feels fine. The sample meets a highly organic mobile phase upon injection which is not its favourite environment and there it precipitates - potentially during a steep gradient. And that’s what I have experienced millions of times – especially when the sample is a protein f. x.
So I would keep an eye with the pressure in relation to each injection of sample – if I were in your shoes

Best Regards