Ecochart lichrospher 100: Back pressure

[Nour]

I am using a new Ecochart glass column lichrospher RP18 125-3, 5µm, the mobile phase is 14:86 ACN:Buffer (phosphate buffer pH7 and as ion pairing 0,5g/L of 1-hexan sulfonic acid sodium salt, flow rate 0,9 ml/min. Isocratic mode.
the samples are made up in mobile phase.

My trouble is that the pressure increases with the injection of the samples (the normal pressure is about 143 bar). I can not validate the method because each time i have an over pressure in the system.

I used two new columns now and the pressure becomes very high after couple of injections (about 5 or 8 injections).

some ideas why this occurs and how I can flush the columns?

[Mattias]

Do you premix the acetonitrile and the buffer, or do you let the instrument do it?

If you do not premix and use a low-pressure mixing pump - I am not surprised that you get high pressure (especially if you use an Agilent quarternary pump).

Another classic problem is particles in the sample - filtrate!

[Bruce Hamilton]

The most likely cause is that a component of the sample is blocking the guard (or main - if no guard is used ) column. Is your sample dissolved in mobile phase, or a similar ratio of CH3CN:H2O?.

Check to ensure all your sample stays in solution when it encounters the mobile phase. If it doesn't change the sample solvent or preparation method. If it does, , then still filter the sample, as suggested by Mattias

With regard to back flushing the column, check the manufacturer's cleaning recommendations to ensure the column can be backflushed, and follow their recommended cleaning procedures. If you are not using a guard column, I would suggest adding one.

If the sample is not the problem, then you may have to filter your mobile phase - it's fairly common to premix ion-pairing mobile phases, such as yours, because the premixed solution can often be stored longer, and any insoluble material will be filtered out.

Ensure the hexane sulphonic acid sodium salt is HPLC grade, but if your mobile phase/sample solvent compatibility s is the problem, you would see pressure build up with multiple blank injections.

Bruce Hamilton

[Nour]

Thank you for your replies!

at first I did not used to premix the mobile phase! but after reading in the forum an interesting subject:
(HAS YOUR SYSTEM BEEN INFECTED) we changed our policy and we now premix the mobile phase to prevent bacterial growth.
we were used to premix the mobile phase only in case when the organic pahse i.e MeOH is used at a percentage less than 10.

I was suspecting the column. Since all the points quoted in your replies were checked:

- the sample, which is a perfusion solution containing only the API and NaCl solution,
was prepared in the mobile phase and no cristalisation was observed.(we pipet 25 ml of the sample and make up to volume 50 ml with mobile phase)
- we also premixed the mobile phase to prevent bacterial growth


we managed to flush and recuperate one column that we used for another test (related compounds)

here again the pressure was not stable (increase after 40 injections)

mobile phase was MeOH (15 volumes) : ACN (5 volumes) : acetate buffer pH 5 (80 volumes)
we run a linear gradient ACN is increased to 55 volmes and buffer to 30 in 20 minutes kept during 3 minutes and then we ge back to the first proportion in 2 minutes and we equilibrate in 10 minutes before the next injection. we didn't observe pressure increase with an other old column.

we will check again the way we flush the column even if it is a normal one:
we replace buffer with water then we move in a linear gradient mode to 70 volumes of ACN a nd 30 volumes of water.
The time of flushing is just enough to run 50 ml of solvent at a flow rate of 0.5ml/min

here are some more details:
we use no guard column
we are using a quaternary pump of waters a new HPLC apparatus
The ion pairin is HPLC grade and purchased from merck