Chromatography Forum

Hi everyone,

I have Agilent 1200 system with quaternary pump G1311 (since April 2008). I am working on methanol: water 1:1, 1.2 ml/min, 20C (BAKER HPLC solvents).
Solvents are degassed, not filtered and used for 1-3 days.

Since some time (around 2 months) I have strange problem: regurarly once a week or once two weeks the pressure becomes too high to do measurments (above 300 bars). What I do is disconecting every part of system up to pump and check the pressure. Every time it looks like the pressure starts to be high on the pump. Therefore, I change the teflon frit inside the pump. It is almost white and has no visual impurities but I change it anyway. Then the problem goes away for one or two weeks and everything goes back again.
More, what I did was changing rubber seals inside the pump and inlet and outlet valves. I am cleaning the glass filters, which I put inside the HPLC solvens bottles.

Therefore, I change the teflon frit inside the pump. It is almost white and has no visual impurities but I change it anyway. Then the problem goes away for one or two weeks and everything goes back again.

The obvious conclusion is that you have small particles clogging that frit. Try filtering your mobile phase through a small (0.35 micron or smaller) filter.

Are you running the same method each time ? What are the column dimensions ? Are you running a gradient or isocratic (you imply iso) ? If you are running a gradient than keep in mind that MeOh and Water hit their max pressure around 50/50 mix. If isocratic only, then you could be accumulating large amounts of retained compounds which are building up and clogging the frit (as Tom implied) or column. Do you flush/clean off your column periodically ? If so, how ? Do you filter your samples ? If so, then are you using a filter that is compatible with the mobile phase ? What are the samples dissolved in ? *The more info you can supply the better the advice will be.

Method is gradient, only methanol and water;
sample is dissolved in mehanol and filtered

phase is not filtered, but as I mentioned I use HPLC Baker solvents; is it not enough if I use directly the solvents? by the way, I use that solvents on the other system (VARIAN, the same method) and nothing strange happens with the pressure;

of course I clean the column, but as I said before the problem is not the column, because if I separate the column off the system, and turn on the system without column, the pressure is still high; I think, the problem is the pump or degasser (if it is possible)

just to illustrate, the usuall presurre for that method (before problems) was around 140-150 bar; and since some time, first I have 140 then it rises up o 300-350 bars

OK, if the system w/o a column shows abnormally high pressure, then try to isolate the source of the problem by moving backward through the flow path, lossening fittings as you go (don't forget the detector's flow cell). You should be able to narrow down where the greatest pressure drop is in the system. Normal backpressure at 1 ml/min with no column in-line should be around 14 bars with 100 % MeOH (or about 8 bars with pure ACN). This value could be different depending on how you have your HPLC plumbed up.

Sounds like something is getting "gummed up" or clogged. Does the pressure drop to zero (0) as soon as you open the prime purge valve ? It should. Has the pump been recently maintained (seals, pistons, cleaning and inspection of parts such as check valve and AIV) ? Once you isolate the source of the pressure drop it should be straighforward.

BTW: Does not sound like the degasser is the problem. If the degasser was plugged (a line), then you would be pulling against a gas which should not cause the liquid pressure to soar. Easiest thing to do is to remove the degasser from the sytem. Temporarily bypass it. Run the solvent lines (w/o frits) directly to the proportioning valve inlet (MCGV).

*There have been cases where someone ran an "inappropriate" solvent through the degasser causing the plastic inside to leach into the mobile phase and cause all kinds of problems down stream (in the pump, A/S, Detector) !